搜索

Analyzing the Properties of Murine Intestinal Mucins by Electrophoresis and Histology
电泳和组织学检查分析小鼠肠黏蛋白的性质   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Specialized secretory cells known as goblet cells in the intestine and respiratory epithelium are responsible for the secretion of mucins. Mucins are large heavily glycosylated proteins and typically have a molecular mass higher than 106 Da. These large proteins are densely substituted with short glycan chains, which have many important functional roles including determining the hydration and viscoelastic properties of the mucus gel that lines and protects the intestinal epithelium. In this protocol, we comprehensively describe the method for extraction of murine mucus and its analysis by agarose gel electrophoresis. Additionally we describe the use of High Iron Diamine-Alcian Blue, Periodic Acid Schiff’s-Alcian Blue and immune–staining methods to identify and differentiate between the different states of glycosylation on these mucin glycoproteins, in particular with a focus on sulphation and sialylation.

Keywords: Sialylation(唾液酸化), Sulphation(硫酸化), Glycosylation(糖基化), Mucin(黏蛋白), Secreted mucin(分泌的黏蛋白), Stored mucin(储存黏蛋白), High Iron Diamine(高铁二胺)

Background

A layer of mucus protects the intestinal epithelium and primarily consists of mucins, water, proteins and inorganic salts. The viscous and gel-like properties of the mucus barrier, which enable it to physically protect and lubricate the mucous membranes, are conferred mainly by mucins. Mucins are large heavily glycosylated proteins and typically have a molecular mass higher than 106 Da. Mucins, however, are predominantly decorated with O-glycan sugars, which accounts for up to 80% of their molecular weight. The diverse site-specific and mucin-specific glycosylation patterns influence the properties of the mucin and therefore the mucus gel. It is well known that mucin glycosylation is altered in infection and disease (Arike et al., 2017; Hasnain et al., 2017). Here we describe methods to assess the amounts of intestinal mucins in murine models and assess the changes in glycosylation with a particular focus on sialylation and sulphation of mucins. Previous methods have not differentiated between mucins isolated from the secreted barrier or those stored within the goblet cells. Methods described here can be employed to assess the changes in secreted or goblet cell-stored mucins in each individual animal. Moreover, using high-iron diamine staining changes in amounts of mucins can also be correlated this with changes in mucin glycosylation.

Materials and Reagents

  1. Pipette tips (Thermo Fisher Scientific, FinntipTM Pipette Tip)
  2. 19.5 gauge needle (BD, catalog number: 305187 )
  3. Needles (BD PrecisionGlideTM Needle)
  4. Nitrocellulose membrane (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 88018 )
  5. BIOMAXTM BML film (Carestream Health, catalog number: 876-1520 )
  6. Microscope slides (Menzel Gläser, SuperFrost® Plus) (VWR, catalog number: 631-9483 )
  7. Cover slips (Fisher Scientific, catalog number: 12-546-2 )
  8. Histology cassettes, moulded lid (ProSciTech, catalog number: RCH40-W )
  9. Biopsy Pads, 100% Reticulated Foam (Trajan Scientific and Medical, catalog number: BPBL )
  10. Polystyrene cube
  11. 10 cm Petri dishes (Corning, catalog number: 430591 )
  12. Transfer pipette, polyethylene (Sigma-Aldrich, catalog number: Z350826-500EA )
  13. Cell scraper (VWR, catalog number: 734-0386 )
  14. D-TubeTM dialyzers (Merck, UK)
  15. C57BL/6 mice (Animal Resource Centre, WA, Australia)
  16. 100% ethanol (Sigma-Aldrich, catalog number: 443611 )
  17. Non-sterile phosphate buffered saline (PBS) (Thermo Fisher Scientific, catalog number: 10010031 )
  18. Urea (VWR, BDH®, catalog number: BDH4602 )
  19. Guanidinium chloride (GuCl) (Sigma-Aldrich, catalog number: G3272 )
  20. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 )
  21. Dithiothrethiol (DTT) (Melford Laboratories, catalog number: MB1015 )
  22. Iodoacetamide (Sigma-Aldrich, catalog number: I6125 )
  23. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  24. Bromophenol blue (Sigma-Aldrich, catalog number: B0126 )
  25. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
  26. Tris base (Roche Diagnostics, catalog number: 10708976001 )
  27. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  28. Sodium citrate (Sigma-Aldrich, catalog number: PHR1416 )
  29. Periodic acid (Sigma-Aldrich, catalog number: P7875 )
  30. Acetic acid (Sigma-Aldrich, catalog number: A9967 )
  31. Sodium metabisulphite (Sigma-Aldrich, catalog number: 31448 )
  32. Schiff’s reagent (Sigma-Aldrich, catalog number: 3952016 )
  33. N,N-dimethyl-p-phenylenediamine (HCl) (Sigma-Aldrich, catalog number: D5004 )
  34. N,N-dimethyl-M-phenylenediamine (HCl)2 (Sigma-Aldrich, catalog number: 219223 )
  35. Iron(III) chloride (FeCl3) (Sigma-Aldrich, catalog number: 157740 )
  36. Alcian blue 8GX (AB) (Sigma-Aldrich, catalog number: A5268 )
  37. Tween-20 (Sigma-Aldrich, catalog number: P1379 )
  38. Skimmed milk powder (Sigma-Aldrich, catalog number: 1443825 )
  39. Odyssey® blocking buffer (LI-COR, catalog number: P/N 927-40000 )
  40. Muc2 antibody: Muc2.3, Rabbit polyclonal antibody (made in house) or H300 Muc2, Rabbit polyclonal antibody (Santa Cruz Biotechnology, catalog number: sc-15334 )
  41. Enhanced chemiluminescence substrate, Western Lightning® Plus-ECL (PerkinElmer, catalog number: NEL105001EA )
  42. IRDye® 800CW Donkey anti-Rabbit IgG (H+L) (LI-COR, catalog number: P/N 925-32213 )
  43. Peroxidase AffiniPure Donkey Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch, catalog number: 711-035-152 )
  44. Alkaline Phosphatase AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch, catalog number: 111-055-003 )
  45. Haematoxylin (Sigma-Aldrich, catalog number: H3136 )
  46. Nitro Blue Tetrazolium (Tablet) (Sigma-Aldrich, catalog number: N5514 )
  47. Wax (MÜNZING, catalog number: CERETAN WE 3501 )
  48. Xylene (Sigma-Aldrich, catalog number: 214736 )
  49. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 484016 )
  50. Pertex mounting medium (MEDITE, catalog number: 41-4012-00 )
  51. 10% neutrally buffered formalin (diluted from CONFIX PURPLE, 10% NBF X5 concentrate) (Australian Biostain, catalog number: ACFP )
  52. Guanidinium chloride (GuCl) reduction buffer (see Recipes)
  53. Loading buffer (see Recipes)
  54. TAE buffer (see Recipes)
  55. 4x SSC buffer (see Recipes)
  56. Periodic acid solution (see Recipes)
  57. Sodium metabisulphite solution (see Recipes)
  58. High Iron Diamine solution (see Recipes)
  59. Alcian blue solution (see Recipes)
  60. TBST buffer (see Recipes)
  61. Milk blocking buffer (see Recipes)

Equipment

  1. Curved tweezers
  2. Pipettes (Eppendorf, model: Research® plus )
  3. Spring scissor (Fine Science Tools, catalog number: 15018-10 )
  4. Forceps (World Precision Instruments, catalog number: 501216 )
  5. 15 ml Falcon conical tube rotor (Beckman Coulter, model: C1015 )
  6. Incubator
  7. Electrophoresis gel tank (Figure 1) (Plaztek Scientific, catalog number: Wini-000 )
  8. Electrophoresis power pack (Figure 1) (Select Bioproducts, model: BioVolt 250V )
  9. Vacuum Blotting Unit with Bio-Rad Blotter (Figure 2) (Bio-Rad Laboratories, model: Model 785 )
  10. Odyssey® CLx Imager (LI-COR, model: Odyssey® CLx )
  11. Micro Spin tissue processor (STP) 120 (Microm UK Ltd.)
  12. Micron Cool Cut HM355S microtome (Microm, model: HM355S )
  13. Microtome blade MX35 (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3052835 )
  14. Water bath (Thermoline Scientific, catalog number: TWB-D-SERIES )
  15. Olympus bright field microscope (Olympus Tokyo, Japan)


    Figure 1. Electrophoresis gel tank (right) and electrophoresis power supply (left) for gel electrophoresis analyses of mucins


    Figure 2. Vacuum Blotting Unit with vacuum blotter chamber (front) and vacuum pump (back)

Software

  1. NIS-Elements software v.3.0 (Nikon, Tokyo, Japan)
  2. Image StudioTM Lite software (LI-COR Biosciences)

Procedure

An outline is provided for all procedures included in this protocol (Figure 3)


Figure 3. Outlines of the procedures described

  1. Biochemical analysis of mucins and mucus
    1. Mice are euthanized using cervical dislocation according to approved ethical guidelines by the host institute in a PC2 laboratory.
    2. Mice are sprayed with 70% ethanol to avoid the contamination of samples with hair.
    3. A mid-line incision is made using scissor in the abdominal cavity.
    4. The intestine is pulled out gently using curved tweezers. Intestines are cut at the end attached to the stomach and the rectum end and dissected out. Small intestine, caecum and colon should be kept separately for analysis.
    5. Two different types of analyses can be conducted; total mucus present in the murine intestine to assess the overall changes in the mucus barrier or the secreted mucus which covers and protects the intestinal epithelium.

      For secreted mucus extraction from mouse intestine
      1. Place the intestine in a 10 cm Petri dish. Gently flush the intestines with PBS, to remove fecal matter, using a syringe with a 19.5 gauge needle through one open end. The amount of PBS will vary depending on the mouse strain and/or the part of intestine that are used. Usually up to 5 ml is used for colon, 7 ml for the small intestine and approximately 3-4 ml for the caecum. This material is discarded unless required for microbiota analyses.
      2. Place the intestine in a new Petri dish and flush the intestine slowly 5 times with 3 M urea (2 ml each flush) to obtain the secreted mucus from the lumen of the intestine. 3 M urea is recommended, as higher concentrations of urea or the use of GuCl would result in disrupting the intestinal epithelial cell layer. The material isolated is viscous; therefore use a plastic transfer pipette to transfer the material to a 15 ml Falcon tube on ice.
      3. Intestinal tissue is processed for histology (as described in Procedure B–histochemical analysis of mucin glycoproteins) before (a small section from the intestine) and after flushing (the whole intestine is rolled) with urea to ensure that only the secreted mucus was isolated and the intestinal crypts remained intact.

      For total mucus extraction from mouse intestine
      1. Place the intestine in a 10 cm Petri dish. Gently flush intestine with PBS, to remove fecal matter, using a syringe with a 19.5 gauge needle through one open end. It is important to ensure that the intestine is only gently flushed; otherwise the secreted mucus layer will be lost. The amount of PBS will vary depending on the strain of mice and/or the part of intestine that are used. Usually up to 5 ml is used for colon, 7 ml for the small intestine and approximately 3-4 ml for the caecum. This material is discarded as it consists of insoluble fecal matter and negligible amounts of secreted mucus (as has been previously investigated by the authors, the faecal mucin matter is not detected via Western blotting).
      2. Cut intestine open longitudinally using a spring scissor. Once open, scrape off the residual mucus, using a cell scraper. This will remove the mucins present in the goblet cells as well as the secreted mucus layer and represents the total intestinal mucus.
      3. The mucus is transferred to a 15 ml Falcon tube and solubilized in 6 M GuCl reduction buffer (see Recipes) at 4 °C overnight by rotation using a Beckman Coulter 15 ml rotator. GuCl is a strong chaotrope and denatures proteins and is recommended for the solubilisation of total mucus isolated from animals. Once solubilized, store sample at -80 °C until required later.

    6. Separation of mucins by agarose gel electrophoresis
      1. The total mucus and or secreted mucus can now be analysed using agarose gel electrophoresis.
      2. Before gel electrophoresis, reduce mucus samples with 50 mM DTT for 2 h at 37 °C to break up the disulphide bonds.
      3. After reduction, add 0.125 M iodoacetamide to the mucus sample and incubate for 30 min at room temperature in the dark to carboxymethylate mucus, to prevent reformation of the disulphide bonds.
      4. Up to 3 ml of the isolated samples in GuCl can then be dialyzed in 3-6 M urea using D-TubeTM dialyzers (Mw cut-off of 14 kDa or below is sufficient). Dialysis to urea is essential as GuCl has a very high salt content.
      5. 10% (v/v) loading buffer (see Recipes) is added in samples before electrophoresis.
      6. Samples are electrophoresed on 0.7-1% (w/v) agarose gels (around 5-7 mm in thickness) in TAE buffer (see Recipes) with 0.1% SDS at 30 V for 15 h (Thornton et al., 2000) (Figure 1).
      7. After electrophoresis, transfer mucins onto a nitrocellulose membrane by vacuum blotting in 4x SSC running buffer (see Recipes) at a pressure of 45-50 mbar using a 785 Bio-Rad Vacuum Blotter (Sheehan and Thornton, 2000) for around 1.5-3 h (Figure 2). To ensure a tight seal, 0.7% agarose can be used to seal the sides of the gel during the vacuum blotting process. Once the entire loading dye has disappeared from the gel, the vacuum blotting process is stopped and the gel is disposed. Note, vacuum blotting consistently results in efficient mucin protein transfer and is the recommended method. The nitrocellulose membrane is carefully removed using tweezers and can be analysed by using three distinct methods (as described in steps A7-A9).

        To determine the changes in the glycosylation of mucins, two separate methods can be utilized which are described in detail below. Periodic Acid Schiff’s-Alcian blue staining can differentiate between acidic and neutral mucins, whereas High Iron Diamine-Alcian Blue staining can be used to identify the changes in mucin sulphation and sialylation.

    7. Glycoprotein detection using PAS staining
      1. Volumes of reagents used in this protocol are dependent on the size of the nitrocellulose membrane and should be sufficient to cover the membrane completely.
      2. Wash nitrocellulose membrane with dH2O.
      3. Incubate nitrocellulose membrane with periodic acid solution (see Recipes) at room temperature for 30 min. Periodic acid will oxidise the vicinal diols in mucin glycoproteins.
      4. Wash membrane thoroughly with dH2O to remove the periodic acid solution.
      5. Nitrocellulose membrane is incubated with sodium metabisulphite solution (see Recipes) for 5 min, the solution is discarded and this step is repeated once more. This step is essential to reduce background staining.
      6. Add Schiff’s reagent to the membrane and incubate for approximately 10-15 min at room temperature. The vicinal diols in mucin glycoproteins exposed by periodic acid will react with the Schiff’s reagent to give a purple-magenta colour.
      7. Rinse membrane with sodium metabisulphite solution, followed by a wash with dH2O to stop the reaction.
      8. Air-dry nitrocellulose membrane before taking scanning using Odyssey® CLx Imager and densitometry analyses (Thornton et al., 1994) (Figure 4).


        Figure 4. Scanned nitrocellulose membrane stained with PAS and anti-Muc2 antibody. Total mucus from the colon was extracted from 4 individual C57BL/6 mice and analysed using 1% agarose gel electrophoresis and transferred to nitrocellulose membrane. Nitrocellulose membrane was stained with PAS (A) or anti-Muc2 antibody (B, H300, from Santa Cruz) and developed using infrared-labelled secondary antibody. Membrane was scanned using the Odyssey® CLx Imager.
        Note: Each individual animal colon yields sufficient mucus for gel electrophoresis analyses–indicates empty lane as negative control.

    8. Detection of sulphated glycoprotein using High Iron Diamine (HID)
      1. Volumes of reagents used in this protocol are dependent on the size of the nitrocellulose membrane and should be sufficient to cover the membrane completely.
      2. Wash nitrocellulose membrane with dH2O.
      3. Incubate nitrocellulose membranes with HID solution (see Recipes) for 18 h at room temperature.
      4. Wash membrane in dH2O and air-dry before scanning using Odyssey® CLx Imager (Hasnain et al., 2017).
    9. Immunodetection of mucin proteins
      1. Volumes of reagents used in this protocol are dependent on the size of the nitrocellulose membrane and should be sufficient to cover the membrane completely.
      2. Wash nitrocellulose membrane briefly in TBST buffer (see Recipes).
      3. Block membrane with milk blocking buffer (see Recipes) or Odyssey blocking buffer for at least 30 min at room temperature (alternatively membrane can be kept in blocking buffer at 4 °C overnight).
      4. Wash membrane 2 times with 15 min incubation for each wash using TBST buffer.
      5. Incubate with anti-Muc2 antibody (diluted in blocking buffer) overnight at 4 °C.
      6. Wash membrane 2 times with 5 min incubation for each wash using TBST buffer.
      7. Incubate with appropriate secondary antibody diluted in blocking buffer for 30 min at room temperature.
      8. For horseradish peroxidase (HRP)-conjugated secondary antibodies, peroxidase conjugated donkey anti-rabbit IgG is incubated with membrane at 1 in 10,000 dilution in TBST for 1 h at room temperature. Membrane is then washed 3 times with 5 min incubation for each wash using TBST buffer. Incubate membrane with enhanced chemiluminescence western detection reagent for approximately 2 min and then washed to remove excess and subsequently expose nitrocellulose membrane to BIOMAXTM BML film in the dark and develop.
      9. For the phosphatase conjugated secondary antibody, phosphatase conjugated goat anti-rabbit IgG is incubated with membrane at 1 in 10,000 dilution in TBST for 1 h at room temperature. After 3 times washes in TBST buffer, membrane is then incubated with nitro-blue tetrazolium (NBT)/5-bromo-4-chloroindol-3yl phosphate substrate for 5 min to develop.
      10. For infrared fluorescent dyes labelled antibodies, IRDye® 800CW donkey anti-rabbit IgG is incubated with membrane at 1 in 2,000 dilution in TBST for 1 h at room temperature. After 3 times washes in TBST buffer, nitrocellulose membranes can be directly scanned at 700 or 800 nm using Odyssey® CLx Imager (Figure 4B).

  2. Histochemical analysis of mucin glycoproteins
    1. ‘Swiss Roll’ technique
      1. Dissect the intestine as described in steps A1-A4 (Figure 5A). Intestine is then cut open longitudinally (Figure 5B) using spring scissors and fecal matter is gently removed by using a P200 pipette tip (Figure 5C). Roll the intestine around a 16 gauge needle with the help of tweezers starting from the rectum end (Figures 5D and 5E). The roll is then pinned onto a small piece of polystyrene using 30 gauge needles (Figure 5F) and placed in fixative solution (4% PFA or 10% neutrally buffered formalin Figure 5G) for 24 h (Reilly and Kirsner, 1965).
    2. Tissue processing
      1. Swiss rolls are transferred to embedding cassettes (Figure 5H) and placed in 70% ethanol.
      2. Process specimens using the Micro Spin Tissue processor (STP) 120. Specimens then go through series of solvents (1 change of 70%, 90%, 95% ethanol, 3 changes of 100% ethanol, 3 changes of xylene and 3 changes of paraffin) for a total of 8 h before being embedded in wax.
      3. Paraffin cell blocks (Figure 5I) can be stored at room temperature until cut to a thickness of 4 µm sample using Micron Cool Cut HM355S microtome with microtome blade MX35.
      4. Sections are floated in a warm water bath (37.4 °C) before being positioned on microscope slides, dried at 37 °C overnight and stored at room temperature until use.


        Figure 5. Mouse intestine dissection and ‘Swiss Roll’ technique for histology. Mouse intestine is dissected (A) followed by opening longitudinally using spring scissor (B) and removing feces using a P200 tip (C). Intestine is rolled around a 16 gauge needle with the help of tweezers (D, E). Swiss roll is secured on polystyrene cube with two needles pin in opposite direction (make sure needle is not placed in tissue) (F) before putting into specimen jar with 10% buffer formalin for fixation (G). Fixed Swiss roll is transferred into histology cassette (H) for paraffin embedding (I).

    3. Periodic Acid Schiff’s-Alcian Blue (PAS-AB) staining
      1. Before staining, slides are rehydrated by going through xylene and ethanol gradient for 5 min in each solution (2 changes of xylene, 2 changes of 100% ethanol, 1 change of 95% and 70% ethanol). Rehydrated slides are placed in PBS for further staining.
      2. Place slides in AB solution for 5 min and then wash briefly in ddH2O. AB stains sulphated and sialylated glycoproteins.
      3. For staining of neutral glycoproteins, incubate the slides with 0.1 M potassium hydroxide in dH2O for 30 min at room temperature.
      4. Place slides in 1% periodic acid solution (see Recipes) for 5 min followed by a thorough wash in ddH2O to remove the acidic solution.
      5. The slides are then placed in Schiff’s reagent for 10 min and washed again in ddH2O.
      6. All slides are washed under running dH2O for 10 min and counterstained with haematoxylin for 30 sec.
      7. Slides are washed in hot ddH2O (warm ddH2O at high power, 30 sec in microwave) for 15 to 30 sec.
      8. Slides are placed in ethanol gradients (from 70% to 100%) and xylene for dehydration, mounted using Pertex mounting medium and sealed with cover slips (Figure 6).


        Figure 6. Pictures of PAS-AB stain in small intestine, proximal colon and distal colon of C57BL/6 mice. Neutral glycoproteins are stained in magenta (red arrow, depicting the glycocalyx), mucins that are both acidic and neutrally charged are stained in purple (blue plus magenta stain, green arrow) and acidic glycoproteins are stained in blue (black arrow). These slides were counterstained with haematoxylin (as described in step B3f). Scale bars = 200 µm (low power), 100 µm (high power).

    4. High Iron Diamine-Alcian blue (HID-AB) staining
      1. Prepare HID solution freshly every time within 4-6 h of use (as described in Recipes).
      2. After the slides are passed through the xylene and ethanol gradient as described in step B3a, slides are incubated with HID for 18 h at room temperature. HID will stain sulphated mucins in black.
      3. Wash slides with ddH2O thoroughly to remove the HID staining solution.
      4. Place slides in AB solution for 30 min (Figure 7) (Spicer, 1965), which will stain sialylated mucins.
      5. No counterstaining is required; therefore slides are mounted as above (described in step B3h).


        Figure 7. HID-AB staining in distal colon of C57BL/6 mice. Black stain indicates sulfomucins dominated area (left panel, red arrow), whereas goblet cells with sialomucins (blue) and mixed sulfo-sialomucins (black and blue colors in the right panel, red arrows). Scale bars = 200 µm (low power), 100 µm (high power).

Data analysis

  1. Densitometry analysis for mucin gel
    1. 2-3 biological samples should be included for quantification from each group.
    2. To quantify the band intensity of mucin gel, Image StudioTM Lite software is used. Multiple regions of interest with the same size are drawn over each mucin bands using the object drawing tool in the software. Intensity of bands is automatically calculated by software as arbitrary number. The number can be used for further comparison between groups.
  2. Quantification of histological staining
    1. Quantification is conducted on blinded samples.
    2. To quantify positive staining in tissue sections after histological staining, multiple pictures (usually 3-4 pictures) from one mouse should be taken. Pictures are taken randomly in areas with longitudinally sectioned crypts using an Olympus bright field microscope with a 40x lens in order to keep sampling process unbiased (although pictures can be taken with any lens, it is essential to keep the sampling procedure consistent). It is important to ensure longitudinally sectioned crypts are quantified, as it limits the variation, artifacts and the possibility of false negative staining.
    3. In each image, positive staining is defined by RGB pixel picking tool in NIS-Elements software v.3.0. Clicking the positive stained area using RGB pixel picking tool enables the software to pick areas that are positive for the exact same staining. This is achieved by defining the actual RGB pixel of the positive stained area. Background staining usually with a slightly lighter color (different pixel) will not be picked up by the software. The percentage of the longitudinally sectioned crypt area occupied by positive staining is calculated (Figure 8) for multiple crypts within each specimen and the average for that specimen determined, and used for statistical analysis.


      Figure 8. Quantification of positive staining by using NIS-Elements software. A. Original picture showing Muc2 positive staining in dark brown color. B. Positive staining was defined by RGB pixel picking tool in NIS-Elements software. For example, to quantify goblet cell volume in a longitudinally sectioned crypt, the percent of positive staining per crypt is calculated as the area covered by positive staining divided by the area of the crypt. Three to five individual crypts are analysed for each sample before taking the average value.

Recipes

  1. Guanidinium chloride (GuCl) reduction buffer
    6 M GuCl dissolved in 0.1 M Tris-HCl buffer with 5 mM EDTA, pH 8.0
  2. Loading buffer
    50% (v/v) glycerol in 10x TAE buffer
    Bromophenol blue
    1% (w/v) SDS
  3. TAE buffer
    40 mM Tris acetate in 1 mM EDTA solution, pH 8.0
  4. 4x SSC buffer
    0.6 M sodium chloride and 60 mM sodium citrate in H2O, pH 7.0
  5. Periodic acid solution
    1% (w/v) periodic acid in 3% (v/v) acetic acid solution
  6. Sodium metabisulphite solution
    0.1% (w/v) sodium metabisulphite in 0.01 M HCl solution
  7. High Iron Diamine solution
    120 mg of N,N-dimethyl-M-phenylenediamine (HCl)2
    20 mg of N,N-dimethyl-p-phenylenediamine (HCl)
    Dissolve in 50 ml of ddH2O
    1.4 ml of 10% FeCl3 added immediately (prepare freshly every time)
  8. Alcian blue solution
    1 g of Alcian blue 8GX power
    100 ml of 3% glacial acetic acid
    Adjust pH to 2.5 using acetic acid
  9. TBST buffer
    150 mM NaCl in10 mM Tris-HCl pH 8.0 with 0.05% Tween-20
    10. Milk blocking buffer
    1% (w/v) skimmed milk powder in TBST buffer

Acknowledgments

A University of Queensland Postdoctoral Fellowship currently supports Sumaira Hasnain. The University Of Queensland Foundation Of Research Excellence Award to Sumaira Hasnain supports Ran Wang. The diamine method was originally described by Spicer (Spicer, 1965) and has been modified for our studies. David Thornton, John Sheehan and Ingemar Carlstedt (Thornton et al., 1994) originally developed the method of assessing mucins in human samples using western blotting on nitrocellulose membrane which was adapted for the animal studies.

References

  1. Arike, L., Holmen-Larsson, J. and Hansson, G. C. (2017). Intestinal Muc2 mucin O-glycosylation is affected by microbiota and regulated by differential expression of glycosyltranferases. Glycobiology 27(4): 318-328.
  2. Hasnain, S. Z., Dawson, P. A., Lourie, R., Hutson, P., Tong, H., Grencis, R. K., McGuckin, M. A. and Thornton, D. J. (2017). Immune-driven alterations in mucin sulphation is an important mediator of Trichuris muris helminth expulsion. PLoS Pathog 13(2): e1006218.
  3. Reilly, R. W. and Kirsner, J. B. (1965). Runt intestinal disease. Lab Invest 14: 102-107.
  4. Sheehan, J. K. and Thornton, D. J. (2000). Heterogeneity and size distribution of gel-forming mucins. Methods Mol Biol 125: 87-96. Spicer, S. S. (1965). Diamine methods for differentialing mucosubstances histochemically. J Histochem Cytochem 13: 211-234.
  5. Spicer, S. S. (1965). Diamine methods for differentialing mucosubstances histochemically. J Histochem Cytochem 13: 211-234.
  6. Thornton, D. J., Carlstedt, I. and Sheehan, J. K. (1994). Identification of glycoproteins on nitrocellulose membranes and gels. Methods Mol Biol 32: 119-128.
  7. Thornton, D. J., Khan, N. and Sheehan, J. K. (2000). Separation and identification of mucins and their glycoforms. Methods Mol Biol 125: 77-85.

简介

在肠和呼吸上皮中称为杯状细胞的专门分泌细胞负责粘蛋白的分泌。 粘蛋白是大的重糖基化蛋白质,通常具有高于10μM的分子量。 这些大蛋白质被短聚糖链密集取代,其具有许多重要的功能作用,包括测定粘液凝胶的水合和粘弹性质,其保护肠上皮。 在该方案中,我们全面地描述了通过琼脂糖凝胶电泳提取小鼠粘液的方法及其分析。 此外,我们描述了使用高铁二胺 - 阿尔星蓝,周期酸席夫氏 - 阿尔辛蓝和免疫染色方法来鉴别和区分这些粘蛋白糖蛋白上糖基化的不同状态,特别是侧重于硫酸化和唾液酸化。
【背景】一层粘液保护肠上皮,主要由粘蛋白,水,蛋白质和无机盐组成。粘液屏障的粘性和凝胶状特性使其能够物理保护和润滑粘膜,主要由粘蛋白赋予。粘蛋白是大的重糖基化蛋白质,通常具有高于10μM的分子量。然而,粘蛋白主要用O-聚糖糖装饰,其占分子量的80%。不同的位点特异性和粘蛋白特异性糖基化模式影响粘蛋白和粘液凝胶的性质。众所周知,粘蛋白糖基化在感染和疾病中发生改变(Arike等人,2017; Hasnain等人,2017)。在这里,我们描述了评估鼠模型中肠粘蛋白量的方法,并评估糖基化的变化,特别关注粘蛋白的唾液酸化和硫酸化。以前的方法没有区分从分泌的屏障或储存在杯状细胞内的粘蛋白。可以使用本文描述的方法来评估每个动物中分泌或杯状细胞储存的粘蛋白的变化。此外,使用高铁二胺染色的粘蛋白量的变化也可以与粘蛋白糖基化的变化相关联。

关键字:唾液酸化, 硫酸化, 糖基化, 黏蛋白, 分泌的黏蛋白, 储存黏蛋白, 高铁二胺

材料和试剂

  1. 移液器提示(Thermo Fisher Scientific,Finntip TM 移液器提示)
  2. 19.5针规(BD,目录号:305187)
  3. 针(BD PrecisionGlide TM 针)
  4. 硝酸纤维素膜(Thermo Fisher Scientific,Thermo Scientific TM,目录号:88018)
  5. BIOMAXTM BML电影(Carestream Health,目录号:876-1520)
  6. 显微镜载玻片(MenzelGläser,SuperFrost Plus)(VWR,目录号:631-9483)
  7. 封面(Fisher Scientific,目录号:12-546-2)
  8. 组织盒,成型盖(ProSciTech,目录号:RCH40-W)
  9. 活检垫,100%网状泡沫(Trajan Scientific and Medical,目录号:BPBL)
  10. 聚苯乙烯立方体
  11. 10厘米培养皿(康宁,目录号:430591)
  12. 转移移液管,聚乙烯(Sigma-Aldrich,目录号:Z350826-500EA)
  13. 电池刮刀(VWR,目录号:734-0386)
  14. D-Tube TM 透析器(Merck,UK)
  15. C57BL / 6小鼠(澳大利亚西澳大利亚动物资源中心)
  16. 100%乙醇(Sigma-Aldrich,目录号:443611)
  17. 非无菌磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,目录号:10010031)
  18. 尿素(VWR,BDH ®,目录号:BDH4602)
  19. 氯化胍(GuCl)(Sigma-Aldrich,目录号:G3272)
  20. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E9884)
  21. 二硫苏糖醇(DTT)(Melford实验室,目录号:MB1015)
  22. 碘乙酰胺(Sigma-Aldrich,目录号:I6125)
  23. 甘油(Sigma-Aldrich,目录号:G5516)
  24. 溴苯酚蓝(Sigma-Aldrich,目录号:B0126)
  25. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L3771)
  26. Tris碱(Roche Diagnostics,目录号:10708976001)
  27. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  28. 柠檬酸钠(Sigma-Aldrich,目录号:PHR1416)
  29. 周期酸(Sigma-Aldrich,目录号:P7875)
  30. 乙酸(Sigma-Aldrich,目录号:A9967)
  31. 焦亚硫酸钠(Sigma-Aldrich,目录号:31448)
  32. 席夫试剂(Sigma-Aldrich,目录号:3952016)
  33. N,N,N-二甲基 - 苯二胺(HCl)(Sigma-Aldrich,目录号:D5004)
  34. N,N,N-二甲基-M-苯二胺(HCl)2(Sigma-Aldrich,目录号:219223)
  35. 氯化铁(III)(FeCl 3)(Sigma-Aldrich,目录号:157740)
  36. 阿尔肯蓝8GX(AB)(Sigma-Aldrich,目录号:A5268)
  37. 吐温-20(Sigma-Aldrich,目录号:P1379)
  38. 脱脂奶粉(Sigma-Aldrich,目录号:1443825)
  39. Odyssey ®阻塞缓冲区(LI-COR,目录号:P / N 927-40000)
  40. Muc2抗体:Muc2.3,兔多克隆抗体(内部制造)或H300 Muc2,兔多克隆抗体(Santa Cruz biotechnology,目录号:sc-15334)
  41. 增强型化学发光底物,Western Lightning Plus / ECL(PerkinElmer,目录号:NEL105001EA)
  42. IRDye ® 800CW驴抗兔IgG(H + L)(LI-COR,目录号:P / N 925-32213)
  43. 过氧化物酶AffiniPure驴抗兔IgG(H + L)(Jackson ImmunoResearch,目录号:711-035-152)
  44. 碱性磷酸酶AffiniPure山羊抗兔IgG(H + L)(Jackson ImmunoResearch,目录号:111-055-003)
  45. 苏木精(Sigma-Aldrich,目录号:H3136)
  46. 硝基蓝四唑(片剂)(Sigma-Aldrich,目录号:N5514)
  47. 蜡(MÜNZING,目录号:CERETAN WE 3501)
  48. 二甲苯(Sigma-Aldrich,目录号:214736)
  49. 氢氧化钾(KOH)(Sigma-Aldrich,目录号:484016)
  50. Pertex安装介质(MEDITE,目录号:41-4012-00)
  51. 10%中性缓冲福尔马林(从CONFIX PURPLE稀释,10%NBF X5浓缩物稀释)(澳大利亚Biostain,目录号:ACFP)
  52. 氯胍(GuCl)还原缓冲液(参见食谱)
  53. 加载缓冲区(见配方)
  54. TAE缓冲区(见配方)
  55. 4x SSC缓冲液(见配方)
  56. 周期酸溶液(见配方)
  57. 偏亚硫酸氢钠溶液(见配方)
  58. 高铁二胺溶液(见配方)
  59. Alcian蓝色解决方案(见配方)
  60. TBST缓冲区(见配方)
  61. 牛奶阻塞缓冲液(见配方)

设备

  1. 弯曲镊子
  2. 移液器(Eppendorf,型号:Research ® plus)
  3. 弹簧剪(Fine Science Tools,目录号:15018-10)
  4. 镊子(世界精密仪器,目录号:501216)
  5. 15 ml Falcon锥形管转子(Beckman Coulter,型号:C1015)
  6. 孵化器
  7. 电泳凝胶槽(图1)(Plaztek Scientific,目录号:Wini-000)
  8. 电泳电源组(图1)(选择Bioproducts,型号:BioVolt 250V)
  9. (Bio-Rad Laboratories,型号:785型)
  10. Odyssey ® CLx Imager(LI-COR,型号:Odyssey ® CLx)
  11. 微自旋组织处理器(STP)120(Microm UK Ltd.)
  12. Micron Cool Cut HM355S切片机(Microm,型号:HM355S)
  13. Microtome刀片MX35(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:3052835)
  14. 水浴(Thermoline Scientific,目录号:TWB-D-SERIES)
  15. 奥林巴斯明场显微镜(奥林巴斯东京,日本)


    图1.电泳凝胶槽(右)和电泳电源(左侧)用于凝胶电泳分析粘蛋白


    图2.具有真空吸墨室(正面)和真空泵(背面)的真空印迹单元

软件

  1. NIS-Elements软件v.3.0(Nikon,Tokyo,Japan)
  2. Image Studio TM Lite软件(LI-COR Biosciences)

程序

为本协议中包含的所有程序(图3)
提供了一个大纲

图3.描述的程序的大纲

  1. 粘液和粘液的生物化学分析
    1. 根据PC2实验室的宿主机构根据批准的道德准则,使用颈椎脱位使小鼠安乐死。
    2. 用70%乙醇喷雾小鼠以避免毛发样品的污染。
    3. 中线切口用腹部剪刀制成。
    4. 使用弯曲的镊子轻轻地拉出肠。在胃和直肠末端附近切断肠,并将其切开。小肠,盲肠和结肠应分开保存以进行分析。
    5. 可以进行两种不同的分析:存在于小肠中的总粘液以评估覆盖并保护肠上皮的粘液屏障或分泌的粘液的总体变化。

      1. 将肠子放在10厘米的培养皿中。用PBS轻轻冲洗肠道,以便通过一个开口端使用带​​有19.5针的针头的注射器去除粪便。 PBS的量将根据所使用的小鼠应变和/或部分肠而变化。通常使用最多5 ml用于结肠,7 ml用于小肠,大约3-4 ml用于盲肠。这种材料被丢弃,除非微生物群分析需要。
      2. 将肠子放入新的陪替氏培养皿中,用3M尿素(每次冲洗2ml)缓慢冲洗肠道5次,以从肠腔中获得分泌的粘液。推荐使用3M尿素,因为较高浓度的尿素或使用GuCl会导致肠上皮细胞层的破坏。分离的材料是粘稠的;因此使用塑料转移移液管将材料转移到冰上的15 ml Falcon管中。
      3. 将肠组织加工成组织学(如方法B - 粘蛋白糖蛋白的组织化学分析所述)(在肠的一小部分之间)和用尿素冲洗(整个肠)之后,确保仅分泌分泌的粘液,肠隐窝保持完好。

      1. 将肠子放在10厘米的培养皿中。用PBS轻轻冲洗肠道,以便通过一个开口端使用带​​19.5针规的针头的注射器去除粪便。重要的是确保肠只被轻轻地冲洗;否则分泌的粘液层将会丢失。 PBS的量将根据所使用的小鼠和/或部分肠的应变而变化。通常使用最多5 ml用于结肠,7 ml用于小肠,大约3-4 ml用于盲肠。这种材料被丢弃,因为它由不溶性粪便物质和可忽略量的分泌的粘液组成(如以前由作者研究的,通过Western印迹未检测到粪便粘蛋白物质)。
      2. 使用弹簧剪刀将肠切开纵向。一旦打开,刮掉剩余的粘液,使用细胞刮刀。这将去除存在于杯状细胞以及分泌的粘液层中的粘蛋白并代表总肠粘液。
      3. 将粘液转移到15ml Falcon管中,并使用Beckman Coulter 15ml旋转器通过旋转在4℃下溶解在6M GuCl还原缓冲液(参见食谱)中。 GuCl是一种强烈的离液剂,使蛋白质变性,推荐用于从动物中分离的总粘液的溶解。一旦溶解,将样品储存在-80°C,直到需要。

    6. 通过琼脂糖凝胶电泳分离粘蛋白
      1. 现在可以使用琼脂糖凝胶电泳分析总粘液和/或分泌的粘液。
      2. 在凝胶电泳之前,在37℃下用50mM DTT将粘液样品减少2小时以分解二硫键。
      3. 还原后,向粘液样品中加入0.125M碘乙酰胺,并在室温下在黑暗中孵育30分钟,使其成为羧甲基化粘液,以防止二硫键的重组。
      4. 然后可以使用D-Tube TM透析器(3-6kDa或更低的Mw截止值)在GuCl中最多3ml分离的样品在3-6M尿素中透析。透析尿素是必不可少的,因为GuCl具有非常高的盐含量。
      5. 10%(v / v)加载缓冲液(见食谱)在电泳前加入样品
      6. 将样品在含有0.1%SDS的TAE缓冲液(参见食谱)中,在30V下在0.7-1%(w / v)琼脂糖凝胶(约5-7mm厚度)上电泳15小时(Thornton等, / em>,2000)(图1)。
      7. 电泳后,使用785 Bio-Rad真空印刷机(Sheehan and Thornton,2000),在4×SSC运行缓冲液(参见食谱)中以45-50毫巴的压力通过真空吸印将粘蛋白转移到硝酸纤维素膜上约1.5-3小时(图2)。为了确保密封,在真空吸印过程中可以使用0.7%的琼脂糖来密封凝胶的两侧。一旦整个加载染料从凝胶中消失,就停止真空吸印过程并且凝胶被处理。注意,真空吸印一贯导致有效的粘蛋白蛋白转移,是推荐的方法。使用镊子小心地去除硝酸纤维素膜,并且可以通过使用三种不同的方法进行分析(如步骤A7-A9所述)。

        为了确定粘蛋白糖基化的变化,可以使用下面详细描述的两种分开的方法。周期酸Schiff's-Alcian蓝染色可以区分酸性和中性粘蛋白,而高铁二胺 - 阿尔西蓝染色可用于鉴定粘蛋白硫酸化和唾液酸化的变化。

    7. 使用PAS染色检测糖蛋白
      1. 本方案中使用的试剂体积取决于硝酸纤维素膜的尺寸,应足以完全覆盖膜。
      2. 用dH 2 O洗涤硝酸纤维素膜。
      3. 在室温下将硝酸纤维素膜与高碘酸溶液孵育30分钟。周期酸将氧化粘蛋白糖蛋白中的邻位二醇。
      4. 用dH 2 O 3彻底清洗膜以除去高碘酸溶液。
      5. 将硝酸纤维素膜与偏亚硫酸氢钠溶液(参见食谱)一起孵育5分钟,弃去溶液,再重复该步骤。这一步对于减少背景染色至关重要。
      6. 将Schiff试剂加入到膜中,并在室温下孵育约10-15分钟。通过高碘酸暴露的粘蛋白糖蛋白中的邻位二醇将与希夫试剂反应,产生紫红色的颜色。
      7. 用偏亚硫酸氢钠溶液冲洗膜,然后用dH 2 O 3洗涤以停止反应。
      8. 在使用Odyssey ® CLx Imager和光密度分析(Thornton等人,1994)(图4)进行扫描之前,使用空气干燥的硝酸纤维素膜。


        图4.用PAS和抗Muc2抗体染色的扫描的硝酸纤维素膜从4只单独的C57BL / 6小鼠中提取来自结肠的总粘液,并使用1%琼脂糖凝胶电泳分析并转移到硝酸纤维素膜上。硝酸纤维素膜用PAS(A)或抗Muc2抗体(B,H300,来自Santa Cruz)染色,并使用红外标记的二抗展开。使用Odyssey ® CLx Imager扫描膜。
        注意:每个单独的动物结肠产生足够的粘液用于凝胶电泳分析 - 表明空泳道为阴性对照。

    8. 使用高铁二胺(HID)检测硫酸化糖蛋白
      1. 本方案中使用的试剂体积取决于硝酸纤维素膜的尺寸,应足以完全覆盖膜。
      2. 用dH 2 O洗涤硝酸纤维素膜。
      3. 在室温下孵育硝酸纤维素膜与HID溶液(见食谱)18小时
      4. 在使用Odyssey CLx Imager(Hasnain等人,2017年)进行扫描之前,在dH 2 O中清洗膜并进行空气干燥。 >
    9. 粘蛋白蛋白免疫检测
      1. 本方案中使用的试剂体积取决于硝酸纤维素膜的尺寸,应足以完全覆盖膜。
      2. 在TBST缓冲液中短暂洗涤硝酸纤维素膜(见食谱)。
      3. 在室温下用阻滞缓冲液(参见食谱)或奥德赛封闭缓冲液至少30分钟的膜(或者膜可以在4℃下保持封闭缓冲液过夜)。
      4. 洗涤膜2次,每次用TBST缓冲液洗涤15分钟
      5. 与抗Muc2抗体(在封闭缓冲液中稀释)一起在4℃下孵育
      6. 使用TBST缓冲液,每次洗涤5分钟,洗涤膜2次
      7. 与在封闭缓冲液中稀释的合适的二次抗体在室温下孵育30分钟
      8. 对于辣根过氧化物酶(HRP)共轭二级抗体,将过氧化物酶缀合的驴抗兔IgG与TBST在1×10,000稀释液中在室温下孵育1小时。然后使用TBST缓冲液将膜洗涤3次,每次洗涤5分钟。用增强的化学发光西方检测试剂孵育膜约2分钟,然后洗涤以除去过量的残留物,随后在黑暗中将硝酸纤维素膜暴露于BIOMAX< B> BML膜。
      9. 对于磷酸酶缀合的第二抗体,将磷酸酶缀合的山羊抗兔IgG与TBST在1 / 10,000稀释液中在室温下孵育1小时。在TBST缓冲液洗涤3次后,然后将膜与硝基蓝四氮唑(NBT)/ 5-溴-4-氯吲哚-3-基磷酸盐底物孵育5分钟,开发。
      10. 对于红外荧光染料标记的抗体,IRDye800CW驴抗兔IgG在室温下用TBST在1 / 2,000稀释液中与膜温育1小时。在TBST缓冲液中洗涤3次后,硝酸纤维素膜可以使用OdysseyCLx成像仪直接在700或800nm进行扫描(图4B)。

  2. 粘蛋白糖蛋白的组织化学分析
    1. “瑞士卷”技术
      1. 如步骤A1-A4(图5A)所述解剖肠。然后使用弹簧剪刀纵向切开肠(图5B),并通过使用P200移液管针头缓缓除去粪便物质(图5C)。在直肠末端从镊子的帮助下将肠围绕16号针头(图5D和5E)。然后使用30号针(图5F)将卷固定在小块聚苯乙烯上,并放置在固定溶液(4%PFA或10%中性缓冲福尔马林图5G)中24小时(Reilly和Kirsner,1965)。 />
    2. 组织加工
      1. 将瑞士卷转移到嵌入盒(图5H)中并置于70%乙醇中。
      2. 使用微自旋组织处理器(STP)的过程样品120.样品然后经过一系列溶剂(1个变化为70%,90%,95%乙醇,3个100%乙醇变化,3个二甲苯变化和3个变化的石蜡)在嵌入蜡之前共8小时。
      3. 石蜡细胞块(图5I)可以在室温下储存,直到用切片刀片MX35的Micron Cool Cut HM355S切片机切割成4μm样品的厚度。
      4. 将切片在温水浴(37.4℃)中漂浮,然后放置在显微镜载玻片上,在37℃下干燥过夜,并在室温下储存直至使用。


        图5.小鼠肠切除术和“Swiss Roll”技术用于组织学小鼠肠被解剖(A),然后使用弹簧剪刀(B)纵向打开,并使用P200尖端(C)去除粪便。在镊子(D,E)的帮助下,肠子围绕16号针头卷绕。将瑞士卷固定在聚苯乙烯立方体上,两个针针相反方向(确保针不放置在组织中)(F),然后放入具有10%缓冲福尔马林固定的样品瓶(G)中。固定的瑞士卷被转移到用于石蜡包埋的组织学盒(H)中(I)
    3. 周期酸Schiff's-Alcian Blue(PAS-AB)染色
      1. 染色前,通过二甲苯和乙醇在各溶液中梯度洗脱5分钟(2次二甲苯,2次100%乙醇变化,1次95%,70%乙醇),使载玻片再水化。将再水化的载玻片置于PBS中以进一步染色。
      2. 将载玻片在AB溶液中放置5分钟,然后在ddH 2 O中短暂洗涤。 AB染色硫酸化和唾液酸化糖蛋白。
      3. 对于中性糖蛋白的染色,在室温下将载玻片与0.1M氢氧化钾在dH 2 O中孵育30分钟。
      4. 将载玻片置于1%的高碘酸溶液(参见食谱)中5分钟,然后在ddH 2 O中彻底洗涤以除去酸性溶液。
      5. 然后将载玻片放入希夫试剂中10分钟,再次在ddH 2 O中洗涤。
      6. 将所有载玻片在运行的dH 2 O下洗涤10分钟,并用苏木精复染30秒。
      7. 将滑块在热的ddH 2 O(加热的ddH 2 O,高功率,微波30秒)下洗涤15至30秒。
      8. 将幻灯片置于乙醇梯度(70%至100%)中,并用二甲苯脱水,使用Pertex安装介质安装并用盖玻片密封(图6)。


        图6. C57BL / 6小鼠的小肠,近端结肠和远端结肠中PAS-AB染色的图片中性糖蛋白以品红色(红色箭头,描绘糖萼)染色,两者均为粘蛋白酸性和中性电荷以紫色(蓝色加洋红色染色,绿色箭头)染色,酸性糖蛋白以蓝色(黑色箭头)染色。这些载玻片用苏木精复染(如步骤B3f所述)。比例尺=200μm(低功率),100μm(大功率)。

    4. 高铁二胺 - 阿尔辛蓝(HID-AB)染色
      1. 每次使用4-6小时内,每次准备HID溶液(如食谱中所述)。
      2. 将载玻片通过二甲苯和乙醇梯度,如步骤B3a所述,将载玻片与HID在室温下孵育18小时。 HID会使黑色的硫酸化粘蛋白染色。
      3. 用ddH 2 O彻底清洗载玻片以除去HID染色溶液。
      4. 将载玻片在AB溶液中放置30分钟(图7)(Spicer,1965),其将染色唾液酸化的粘蛋白。
      5. 不需要复染;因此,如上所述安装幻灯片(在步骤B3h中描述)。


        图7. C57BL / 6小鼠远端结肠中的HID-AB染色黑色染色指示磺基琥珀酸主导区域(左图,红色箭头),而具有唾液酸蛋白(蓝色)和混合磺基 - 唾液酸蛋白的杯状细胞(黑色和蓝色的颜色在右面板,红色箭头)。比例尺=200μm(低功率),100μm(大功率)。

数据分析

  1. 粘蛋白凝胶的光密度分析
    1. 应包括2-3个生物样品用于每组的定量。
    2. 为了量化粘蛋白凝胶的带强度,使用Image Studio TM Lite软件。使用软件中的对象绘图工具在每个粘蛋白条带上绘制具有相同尺寸的多个感兴趣的区域。频带强度由软件自动计算为任意数。该数字可用于组之间的进一步比较。
  2. 组织学染色的定量
    1. 对盲样进行定量。
    2. 为了在组织学染色后定量组织切片中的阳性染色,应采取一只小鼠的多张照片(通常3-4张照片)。使用奥林巴斯明视野显微镜(40x镜头)将图像随机地放置在具有纵截面隐藏区域,以保持采样过程无偏差(尽管可以使用任何镜头拍摄照片,但必须保持采样过程一致)。重要的是确保纵向切片的隐窝被量化,因为它限制了变异,伪像和假阴性染色的可能性。
    3. 在每个图像中,NIS-Elements软件v.3.0中的RGB像素拾取工具定义了正染色。使用RGB像素拾取工具点击正面染色区域,软件可以选择对于完全相同染色呈阳性的区域。这通过定义正染色区域的实际RGB像素来实现。背景染色通常使用稍微较浅的颜色(不同像素)不会被软件拾取。对每个样本中的多个隐窝计算正面染色所占据的纵剖面隐藏区域的百分比(图8),并确定该样本的平均值,并用于统计分析。


      图8.使用NIS-Elements软件定量阳性染色 A.原始图片显示深棕色的Muc2阳性染色。 B.阳性染色由NIS-Elements软件中的RGB像素拾取工具定义。例如,为了量化纵断面隐窝中的杯状细胞体积,计算每个隐窝的阳性染色百分比,其中阳性染色所覆盖的面积除以隐窝的面积。在取平均值之前,分析每个样本的三到五个个体隐窝。

食谱

  1. 氯胍(GuCl)还原缓冲液
    6M GuCl溶解在具有5mM EDTA,pH 8.0的0.1M Tris-HCl缓冲液中
  2. 加载缓冲区
    50%(v / v)甘油在10倍TAE缓冲液中 溴酚蓝
    1%(w / v)SDS
  3. TAE缓冲区
    40mM Tris乙酸盐在1mM EDTA溶液中,pH8.0
  4. 4x SSC缓冲区
    0.6M氯化钠和60mM柠檬酸钠在H 2 O,pH 7.0中
  5. 周期酸溶液
    1%(w / v)高碘酸在3%(v / v)乙酸溶液中
  6. 偏亚硫酸氢钠溶液
    0.1%(w / v)偏亚硫酸氢钠在0.01M HCl溶液中
  7. 高铁二胺溶液
    120mg N,N'-二乙基间苯二胺(HCl)2
    20mg N,N'-二甲基 - 苯二胺(HCl)
    溶于50ml的ddH 2 O - / - 立即添加1.4ml 10%FeCl 3(新鲜每次准备)
  8. Alcian蓝色解决方案
    1克Alcian蓝8GX电源
    100ml 3%冰醋酸
    使用乙酸将pH调节至2.5
  9. TBST缓冲区
    150mM NaCl,10mM Tris-HCl pH 8.0,含0.05%Tween-20 牛奶阻塞缓冲器
    TBST缓冲液中1%(w / v)脱脂奶粉

致谢

昆士兰大学博士后研究员目前支持Sumaira Hasnain。昆士兰大学研究优秀奖基金会Sumaira Hasnain支持冉王。二胺方法最初由Spicer(Spicer,1965)描述,并已被修改用于我们的研究。 David Thornton,John Sheehan和Ingemar Carlstedt(Thornton等人,1994)最初开发了使用适于动物研究的硝酸纤维素膜上的蛋白质印迹来评估人类样品中的粘蛋白的方法。

参考

  1. Arike,L.,Holmen-Larsson,J.and Hansson,GC(2017)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/28122822 “目标=”_空白“>肠道Muc2粘蛋白O-糖基化受微生物群落的影响,并受糖基转移酶的差异表达调控。糖生物学 27(4):318-328。
  2. Hasnain,SZ,Dawson,PA,Lourie,R.,Hutson,P.,Tong,H.,Grencis,RK,McGuckin,MA和Thornton,DJ(2017)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/28192541”target =“_ blank”>粘蛋白硫酸化的免疫驱动改变是三叉戟蠕虫驱除的重要介质。 PLoS Pathog 13(2):e1006218。
  3. Reilly,RW和Kirsner,JB(1965)。  Runt肠道疾病。实验室投资 14:102-107。
  4. Sheehan,JK and Thornton,DJ(2000)。  异质性和凝胶形成粘蛋白的大小分布。 Methods Mol Biol 125:87-96。 Spicer,S. S.(1965)。组胺化学差异粘膜状态的二胺方法。 J Histochem Cytochem 13:211-234。
  5. Spicer,SS(1965)。用于区分粘液状态的二胺方法组织化学。 J Histochem Cytochem 13:211-234。
  6. Thornton,DJ,Carlstedt,I. and Sheehan,JK(1994)。  鉴定硝酸纤维素膜和凝胶上的糖蛋白。方法Mol Biol 32:119-128。
  7. Thornton,DJ,Khan,N. and Sheehan,JK(2000)。  分离和鉴定粘蛋白及其糖型。方法Mol Biol 125:77-85。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wang, R. and Hasnain, S. Z. (2017). Analyzing the Properties of Murine Intestinal Mucins by Electrophoresis and Histology. Bio-protocol 7(14): e2394. DOI: 10.21769/BioProtoc.2394.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。