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Histochemical Staining of Collagen and Identification of Its Subtypes by Picrosirius Red Dye in Mouse Reproductive Tissues
使用天狼星红染料对小鼠生殖组织中胶原蛋白进行组织化学染色并鉴定其亚型   

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Abstract

Collagen is one of the foremost components of tissue extracellular matrix (ECM). It provides strength, elasticity and architecture to the tissue enabling it to bear the wear and tear from external factors like physical stress as well as internal stress factors like inflammation or other pathological conditions. During normal pregnancy or pregnancy related pathological conditions like preterm premature rupture of membranes (PPROM), collagen of the fetal membrane undergoes dynamic remodeling defining biochemical properties of the fetal membrane. The protocol in this article describes the histochemical method to stain total collagen by Picrosirius red stain which is a simple, quick and reliable method. This protocol can be used on paraformaldehyde (PFA) and formaldehyde fixed paraffin embedded tissue sections. We further describe the staining and distribution of collagen in different mouse reproductive tissues and also demonstrate how this technique in combination with polarization microscopy is useful to detect the distribution of different subtypes of collagen.

Keywords: Collagen(胶原), Histochemical staining(组织化学染色), Picrosirius red(天狼星红), Reproductive tissues(生殖组织), Birefringence(双折射), Polarization(极化)

Background

Collagen is the principal load-bearing polymer in all connective tissues ranging from skin to bone. Collagen networks strongly stiffen when a mechanical force is applied, thus preventing excessive deformation of the tissue. There are 16 types of collagen, of which the type I, II, and III nearly comprise the 80% of the collagen in the body that are packed together to form long thin fibrils. Collagen type IV forms a two-dimensional reticulum; while several other collagen types are associated with fibril-type collagen, linking them to each other or to other matrix components. These collagens along with the other components of the extracellular matrix (ECM) undergo constant remodeling to provide required biochemical properties like tensile strength and elasticity. This unique attribute of collagen is one of the influencing factors of the stability of reproductive tissues and its dysregulation can lead to adverse events such as abnormal placentation, rupture of membranes (Hampson et al., 1997; Marpaung, 2016) and pathological conditions of the reproductive tract, such as endometriosis (Shimizu and Hokano, 1990) etc.

In tissues, the basement membrane is rich in collagen, in addition it is found in the stroma and lining the connective tissues. Physical, mechanical or chemical damage of a tissue or organ would lead to disruption of collagen deposition, and organization. Hence, assessing the patterns of collagen distribution would provide us an idea about the tensile strength of the tissue/organs. Any alterations from normal patterns of collagen distribution would imply tissue damage. In this study, chorio-decidual tissue has been used as a model basement membrane. Changes in the biochemical properties of this feto-maternal membrane during pregnancy and various pathological conditions lead to its preterm rupture (Sebire, 2001; Fujimoto et al., 2002; Wang et al., 2004; Vega Sánchez et al., 2004; Surve et al., 2016).

Sirius red is a histology stain used to mark total collagen as well as differentiate between varying collagen types for evaluation of collagen distribution in tissues. The sulphonic acid group of Sirius red reacts with basic amino groups of lysine and hydroxylysine and guanidine group of arginine (present in the collagen molecule (Junqueira et al., 1979)). Thereby, being an anionic dye, it attaches to all the varying types of collagen isoforms. In bright field, collagen appears as bundles of pink to red fibers which get disturbed in pathological conditions. The same larger collagen fibers under polarized light appear bright yellow to orange and the thinner ones, including reticular fibers, look green. This birefringence or double refraction, whereby incident light is split by polarization into two different paths, is highly specific for collagen. The amount of polarized light absorbed by the Sirius red dye stringently depends on the orientation of the collagen bundles enabling to differentiate different collagen types (Junqueira et al., 1979; Lattouf et al., 2014). This method is very simple, quick, economic and reliable in comparison with other commonly used staining methods for collagen.

Materials and Reagents

  1. Glass coverslips (HiMedia Laboratories, catalog number: CG108 )
  2. Glass slides (size, 76 x 26 mm) (HiMedia Laboratories, catalog number: CG029 )
  3. Paraffin mold and embedding cassette (Simport, catalog number: M490-2 )
  4. Mice strain: C57BL/6 Black (Experimental Animal Facility, ICMR-National Institute of Research in Reproductive Health)
  5. Distilled water (D/W)
  6. Formaldehyde (Sigma-Aldrich, catalog number: F8775 )
  7. Xylene (Merck, catalog number: 1086342500 )
  8. Methanol (Merck, catalog number: 1070182521 )
  9. Ethanol (Merck, catalog number: 1085430250 )
  10. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
  11. Sodium phosphate monobasic (NaH2PO4)
  12. Sodium phosphate dibasic (Na2HPO4)
  13. Sodium chloride (NaCl)
  14. Potassium chloride (KCl)
  15. Potassium phosphate monobasic (KH2PO4)
  16. Paraffin wax (Merck, catalog number: 1073371000 )
  17. Poly-lysine (Sigma-Aldrich, catalog number: P8920 )
  18. Picric acid (Fisher Scientific, catalog number: 13205 )
  19. Direct Red 80 (Sigma-Aldrich, catalog number: 365548 )
  20. Glacial acetic acid (CH3COOH) (Merck, catalog number: 1.93002 )
  21. Haematoxylin (C.I. 75290) (EMD Millipore, catalog number: 104302 )
  22. Iron(III) chloride (ferric chloride) (Merck, catalog number: 803945 )
  23. Sodium bicarbonate (Merck, catalog number: 106329 )
  24. Hydrochloric acid (37%) (Merck, catalog number: 1.93001 )
  25. D.P.X. mountant liquid (HiMedia Laboratories, catalog number: GRM655 )
  26. Weigert’s haematoxylin solution (see Recipes)
  27. 4% paraformaldehyde (PFA) (see Recipes)
  28. 10% formaldehyde (see Recipes)
  29. Phosphate buffered saline (PBS) (see Recipes)
  30. Poly-lysine coated glass slides (see Recipes)
  31. Picrosirius red solution (see Recipes)
  32. Acidified water (see Recipes)

Equipment

  1. Coplin jar (Thermo Scientific, catalog number: 107 )
  2. Bright field microscope (Leica, model: Leica DMi8 )
  3. Polarization microscope (Leica, model: Leica DMi8 )
    Note: Items 2 and 3 are the same microscope. For polarization applications, a polarizer (Leica Microsystems, Germany) is placed in the light path of the bright field microscope.
  4. Hot plate (LED Digital, Lab Depot, model: MS7-H550-S )
  5. Microtome (Leica, model: Leica RM2255 )

Procedure

  1. Sacrifice mice by cervical dislocation as per the institute ethical guidelines and dissect it to collect the respective tissues. Briefly, following euthanization, make a 1.5-cm midline incision in the lower abdomen and collect different reproductive tissues. To obtain chorio-decidua, incise the bicornuate uterine horns longitudinally along the anti-mesenteric border and collect the fetal membrane.
  2. Fix the tissue overnight at 4 °C in 10% buffered formaldehyde or 4% PFA.
  3. Wash 5 times in PBS (see Recipes) and dehydrate in methanol gradient series (30%, 50%, 70%, 90%, 100% 30 min each) and xylene till tissue gets translucent.
  4. Add molten paraffin (heated at 60 °C on a hot plate) in xylene (v/v, 1:1) and incubate at room temperature for 15 min.
  5. Add molten wax to the tissues and incubate at 60 °C for 15 min. Change the wax and leave the tissues in wax at room temperature.
  6. Next day melt the wax containing the tissue and give one more change of wax and the final incubation lasts for 15 min. For large tissue, the incubation time in wax may be increased to 30-45 min. The timing has to be empirically determined to suit the tissue type.
  7. Pour molten paraffin in the mold and place the tissue in the desired orientation (placing it vertically) and overlay with embedding cassette on it. Leave it overnight at room temperature to completely solidify.
  8. De-mold paraffin embedded tissue along with embedding cassette and section according to standard histopathology protocols using a microtome.
  9. Collect the sections on a poly-lysine coated slide (see Recipes) and let it dry overnight at 37 °C.
  10. Warm the sections on a hot plate (at 60 °C) and quickly dip in xylene containing Coplin jar.
  11. De-paraffinize the sections in xylene twice for 30 min each. Ensure that the sections are nearly translucent.
  12. Dip the slides through either methanol or ethanol gradient series (100%, 90%, 70%, 50%, 30%, D/W 5 min each).
  13. Stain nuclei with haematoxylin (see Recipes) for 5-10 min.
  14. Wash the slides in running tap water for 2 min.
  15. Put the slides in Picrosirius red (see Recipes) containing Coplin jar for 1 h.
  16. Wash the slides twice with fresh acidified water (see Recipes) (5 min each).
  17. Wash the slides in D/W and dehydrate the sections through either methanol or ethanol gradients (50%, 70%, 90% for 5 min each).
  18. Give three changes of 100% ethanol (5 min each).
  19. Incubate in xylene for 30 min (two changes).
  20. Mount in a D.P.X. and let it dry overnight.
  21. Observe under a bright field or polarized microscope. Images can be captured using a routine digital camera or a CCD.

Data analysis

  1. Haematoxylin stains cell nucleus violet in color. However, cell nuclei may appear black or brown or grey. Under a bright-field microscope, collagen appears pink to red in color on a pale yellow background of cytoplasm as a result of staining with Sirius red dye. The Picrosirius red staining not only provides information about collagen distribution in a tissue sample, but also detects bundles of collagen fibers in tissue sections. If one is studying disruption of collagen fibers leading to interruption of bundle formation, staining of collagen by this method can be of great value.
  2. Using Picrosirius red staining, we could demonstrate varying distribution of collagen in mouse reproductive tissues (Figure 1). To demonstrate how this technique is useful to detect collagen degradation, mouse chorio-decidua/amnion was treated with membrane vesicles (MVs) from group B Streptococcus (GBS) which has collagenase activity (Surve et al., 2016). In the normal tissues, bundles of collagen are seen in the chorion and amnion. However, this is fragmented upon treatment with GBS MV. The fragmentation and degradation are clearly visible in the GBS MV treated tissues (Figure 2).


    Figure 1. Collagen staining by Picrosirius red dye in various mouse reproductive tissues and fetal membrane. Red to pink is collagen stain. Yellow is cytoplasm. Scale bars = 50 µm.


    Figure 2. Collagen degradation in the mouse amnion (A) and chorion (C) in response to membrane vesicles (MVs) from GBS. In vivo data are from mouse injected with GBS MVs in the amniotic sacs on E16.5. Ex vivo are chorio-amnion incubated with GBS MVs for 16h. Arrows show collagen degradation. Scale bars = 50 µm.

  3. We utilized the birefringence property of the different forms of collagen to demonstrate their distribution in various mouse reproductive tissues by polarization microscopy (Figure 3). Under polarization microscope, collagen I appears yellow-red (white arrowhead), collagen III appears green (blue arrowhead) and collagen IV shows weak to no birefringence (white outlined black arrow head) (Montes and Junqueira, 1991). A summary of these findings is shown in Table 1.


    Figure 3. Detection of collagen subtypes by polarization microscopy of Picrosirius red dye stained mouse tissues. Collagen I is yellow-red (white arrow head), collagen III is green (Blue arrow head), collagen IV is weak to no birefringence (white outlined black arrow head). Scale bars = 50 µm.

    Table 1. Distribution of collagen in mouse reproductive tissues and in pregnancy as detected by Picrosirius red dye staining coupled with polarization microscopy

Notes

  1. The staining of collagen fibers and its distribution are fairly constant from day to day basis. The staining is reproducible with varying tissues with the same protocol.
  2. Even if the tissue looks stained in shorter time, it should not be taken out before 1 h.
  3. No differences in collagen staining were observed in fresh tissue sections vs. those that were stored at room temperature for more than 2-3 months. Sections that were stained earlier (2 months) with Picrosirirus red demonstrated similar colors and intensity.
  4. Birefringence did not alter upon storage of stained sections. The intensity of birefringence was similar in fresh sections vs. those that were stained and stored at room temperature for more than 2-3 months.

Recipes

  1. Weigert’s haematoxylin solution
    1. Prepare Haematoxylin stock solution
      1.0 g Haematoxylin
      100.0 ml 96% ethanol
      Mix it properly and keep mixture for 1 week for maturation at room temperature
    2. Prepare iron(III) chloride stock solution
      1.16 g iron(III) chloride
      1.0 ml HCl (25%)
      99.0 ml distilled water
      Dissolve it thoroughly
    3. Combine haematoxylin stock solution with iron(III) chloride stock solution in 1:1 ratio to obtain Weigert’s haematoxylin dye solution. Mix it thoroughly prior to use
  2. 10% neutral buffered formalin
    0.4 g NaH2PO4
    0.65 g Na2HPO4
    90 ml distilled water
    10 ml formaldehyde
  3. 4% paraformaldehyde
    4 g paraformaldehyde
    100 ml 1x PBS
    Heat at 65 °C for 5 min till the PFA dissolves
  4. Composition of PBS
    0.8 g NaCl
    0.144 g Na2HPO4
    200 mg KCl
    240 mg KH2PO4
    Add 100 ml D/W
  5. Poly-lysine coated glass slides
    Poly-lysine: 10 µl/coverslip
    Apply on upper surface of clean glass slide evenly
    Allow it to air dry at RT and store it at RT for further use. No need to wash
  6. Picrosirius red solution
    0.1 g Direct red 80
    Saturated aqueous solution of picric acid (1.3 g in 100 ml distilled water)
    Mix it gently till it gets completely dissolved
  7. Acidified water
    1 ml glacial acetic acid in 200 ml distilled water

Acknowledgments

MV Surve and S Bhutda acknowledge fellowship from UGC, Govt. of India, and A Anil acknowledges the same from CSIR, Govt. of India. N Singh acknowledges Junior Research Fellowship from Department of Biotechnology, Govt. of India. Financial assistance from Indian Council of Medical Research (ICMR) and IIT Bombay seed grant to D Modi and A Banerjee, respectively, are also acknowledged. The authors have no conflicts of interest.

References

  1. Fujimoto, T., Parry, S., Urbanek, M., Sammel, M., Macones, G., Kuivaniemi, H., Romero, R. and Strauss, J. F., 3rd (2002). A single nucleotide polymorphism in the matrix metalloproteinase-1 (MMP-1) promoter influences amnion cell MMP-1 expression and risk for preterm premature rupture of the fetal membranes. J Biol Chem 277(8): 6296-6302.
  2. Hampson, V., Liu, D., Billett, E. and Kirk, S. (1997). Amniotic membrane collagen content and type distribution in women with preterm premature rupture of the membranes in pregnancy. Br J Obstet Gynaecol 104(9): 1087-91.
  3. Junqueira, L. C., Bignolas, G. and Brentani, R. R. (1979). Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem J 11(4): 447-455.
  4. Lattouf, R., Younes, R., Lutomski, D., Naaman, N., Godeau, G., Senni, K. and Changotade, S. (2014). Picrosirius red staining: a useful tool to appraise collagen networks in normal and pathological tissues. J Histochem Cytochem 62(10): 751-758.
  5. Marpaung, J. (2016). Association between the thickness of the collagen in the amniotic membrane with the incidence of premature rupture of membranes. Int J Reprod Contracept Obstet Gynecol 5(2):296-299.
  6. Montes G. S. and Junqueira L. C. U. (1991). The use of the picrosirius polarization method for the study of the biopathology of the collagen. Mem Inst Oswaldo Cruz 86(III): 1-11.
  7. Sebire, N. J. (2001). Choriodecidual inflammatory syndrome (CoDIS) is the leading, and under recognised, cause of early preterm delivery and second trimester miscarriage. Med Hypotheses 56(4): 497-500.
  8. Shimizu, K. and Hokano, M. (1990). Effect of loss of mechanical distension on collagen degradation in the mouse uterus. J Anatomy 173: 161-167.
  9. Surve, M. V., Anil, A., Kamath, K. G., Bhutda, S., Sthanam, L. K., Pradhan, A., Srivastava, R., Basu, B., Dutta, S., Sen, S., Modi, D. and Banerjee, A. (2016). Membrane vesicles of group B Streptococcus disrupt feto-maternal barrier leading to preterm Birth. PLoS Pathog 12(9): e1005816.
  10. Vega Sánchez, R., Estrada Gutierrez, G., Cerbulo Vazquez, A., Beltran Montoya, J. and Vadillo Ortega, F. (2004). Characterization of chorioecidual space as an effector molecule-rich environment that induces rupture of fetal membranes during labor. Ginecol Obstet Mex 72: 593-601.
  11. Wang, H., Parry, S., Macones, G., Sammel, M. D., Ferrand, P. E., Kuivaniemi, H., Tromp, G., Halder, I., Shriver, M. D., Romero, R. and Strauss, J. F. 3rd. (2004). Functionally significant SNP MMP8 promoter haplotypes and preterm premature rupture of membranes (PPROM). Hum Mol Genet 13(21): 2659-2669.

简介

胶原蛋白是组织细胞外基质(ECM)最重要的组成部分之一。它为组织提供了强度,弹性和构造,使其能承受来自外部因素的磨损,如身体压力以及炎症或其他病理状况等内部应激因素。在正常妊娠或与妊娠相关的病理状况如胎膜早破(PPROM)期间,胎膜的胶原经历动态重塑,定义胎膜的生化特性。本文中的方案描述了一种简单,快速,可靠的组织化学染色方法,用苦皮红染色法对总胶原进行染色。该协议可用于多聚甲醛(PFA)和甲醛固定石蜡包埋组织切片。我们进一步描述了胶原蛋白在不同的小鼠生殖组织中的染色和分布,并且还展示了这种技术如何与偏振显微镜组合用于检测不同亚型胶原蛋白的分布。

【背景】胶原蛋白是从皮肤到骨头的所有结缔组织中的主要负载聚合物。当施加机械力时,胶原蛋白网络强烈变硬,从而防止组织的过度变形。有16种胶原蛋白,其中I型,II型和III型胶原蛋白几乎占据了人体胶原蛋白的80%,这些胶原蛋白被挤在一起形成细长的原纤维。 IV型胶原蛋白形成二维网状结构;而其他几种胶原蛋白类型与原纤维型胶原蛋白相关,将它们彼此连接或连接到其他基质组分。这些胶原蛋白与细胞外基质(ECM)的其他成分一起经历不断的重塑以提供所需的生物化学性质如拉伸强度和弹性。这种胶原蛋白的独特属性是生殖组织稳定性的影响因素之一,其失调可能导致不良事件,例如胎盘异常,膜破裂(Hampson等人,1997; Marpaung, 2016)和生殖道的病理状况,如子宫内膜异位症(Shimizu和Hokano,1990)等等。

在组织中,基底膜含有丰富的胶原蛋白,此外它还存在于基质和结缔组织衬里中。组织或器官的物理,机械或化学损伤将导致胶原沉积和组织的破坏。因此,评估胶原分布的模式将为我们提供关于组织/器官的抗张强度的想法。正常的胶原蛋白分布模式的任何改变都意味着组织损伤。在这项研究中,绒毛膜蜕膜组织已被用作模型基底膜。胎儿母体膜在妊娠期间的生物化学性质的改变和各种病理状态导致其早期破裂(Sebire,2001; Fujimoto等人,2002; Wang等人, ,2004; VegaSánchez等人,2004; Surve等人,2016)。

天狼星红是一种组织学染色,用于标记总胶原蛋白以及区分各种胶原蛋白类型,以评估组织中的胶原蛋白分布。天狼星红的磺酸基与赖氨酸和羟赖氨酸的碱性氨基以及精氨酸的胍基(存在于胶原分子中(Junqueira等人,1979))反应。因此,作为阴离子染料,它附着于所有不同类型的胶原异构体。在明亮的领域,胶原呈现出粉红色至红色纤维束,在病理条件下受到干扰。偏振光下相同的较大的胶原纤维呈现明亮的黄色至橙色,较薄的包括网状纤维在内呈绿色。这种双折射或双折射,由此入射光通过偏振分裂成两个不同的路径,对胶原蛋白具有高度特异性。由天狼星红染料吸收的偏振光的量严格取决于胶原束的取向,从而能够区分不同的胶原类型(Junqueira等,1979; Lattouf等, em>,2014)。与其他常用的胶原蛋白染色方法相比,该方法简单,快捷,经济可靠。

关键字:胶原, 组织化学染色, 天狼星红, 生殖组织, 双折射, 极化

材料和试剂

  1. 玻璃盖玻片(HiMedia实验室,目录号:CG108)
  2. 玻璃幻灯片(尺寸,76 x 26毫米)(HiMedia实验室,目录号:CG029)
  3. 石蜡模具和包埋盒(Simport,目录号:M490-2)
  4. 小鼠品系:C57BL / 6黑色(实验动物设施,ICMR-National Institute of Research in Reproductive Health)
  5. 蒸馏水(D / W)
  6. 甲醛(Sigma-Aldrich,目录号:F8775)
  7. 二甲苯(Merck,目录号:1086342500)
  8. 甲醇(Merck,目录号:1070182521)
  9. 乙醇(Merck,目录号:1085430250)
  10. 多聚甲醛(Sigma-Aldrich,目录号:P6148)
  11. 磷酸二氢钠(NaH 2 PO 4)
  12. 磷酸二氢钠(Na 2 HPO 4)
  13. 氯化钠(NaCl)
  14. 氯化钾(KCl)
  15. 磷酸二氢钾(KH 2 PO 4)
  16. 石蜡(Merck,目录号:1073371000)
  17. 聚赖氨酸(Sigma-Aldrich,目录号:P8920)
  18. 苦味酸(Fisher Scientific,目录号:13205)
  19. 直接红80(Sigma-Aldrich,目录号:365548)
  20. 冰醋酸(CH 3 3 COOH)(Merck,目录号:1.93002)
  21. 苏木精(C.I. 75290)(EMD Millipore,目录号:104302)
  22. 氯化铁(三氯化铁)(Merck,目录号:803945)
  23. 碳酸氢钠(默克,目录号:106329)
  24. 盐酸(37%)(Merck,目录号:1.93001)
  25. D.P.X. (HiMedia实验室,目录号:GRM655)
  26. Weigert的苏木精溶液(见食谱)
  27. 4%多聚甲醛(PFA)(见食谱)
  28. 10%的甲醛(见食谱)
  29. 磷酸盐缓冲盐水(PBS)(见食谱)
  30. 聚赖氨酸涂层载玻片(见食谱)
  31. Picrosirius红色解决方案(见食谱)
  32. 酸化水(见食谱)

设备

  1. 科普林罐(Thermo Scientific,目录号:107)
  2. 明场显微镜(徕卡,型号:徕卡DMi8)
  3. 偏光显微镜(徕卡,型号:徕卡DMi8)
    注意:项目2和3是相同的显微镜。对于偏振应用,将偏振器(Leica Microsystems,Germany)放置在明场显微镜的光路中。
  4. 热板(LED数字,实验室车库,型号:MS7-H550-S)
  5. 切片机(徕卡,型号:Leica RM2255)

程序

  1. 根据研究所的道德准则,通过颈椎脱臼牺牲小鼠,并解剖它们以收集各自的组织。简单地说,安乐死后,在下腹部做一个1.5厘米的中线切口,收集不同的生殖组织。要获得绒毛膜蜕膜,沿着肠系膜边缘纵向切开双角子宫角,收集胎膜。

  2. 在4°C的10%缓冲甲醛或4%PFA固定组织过夜
  3. 在PBS中清洗5次(见食谱),在甲醇梯度系列(30%,50%,70%,90%,100%30分钟)和二甲苯中脱水,直到组织变得半透明。
  4. 在二甲苯(v / v,1:1)中加入熔融石蜡(在热板上60℃加热)并在室温下孵育15分钟。
  5. 将熔融蜡添加到组织中,并在60℃下孵育15分钟。改变蜡,并在室温下将组织留在蜡中。
  6. 第二天融化含有组织的蜡,再给蜡一次,最后的孵化持续15分钟。对于大的组织,蜡中的孵育时间可以增加到30-45分钟。时间必须根据经验确定以适应组织类型。
  7. 将熔化的石蜡倒入模具中,并将组织放置在所需的方向(垂直放置),并用包埋盒覆盖。在室温下放置一夜,使其完全固化。
  8. 根据标准组织病理学方案,使用切片机,将石蜡包埋的组织与包埋盒和切片一起脱模。
  9. 收集聚赖氨酸涂层载玻片上的部分(见食谱),让它在37℃干燥过夜。
  10. 在热板上(60°C)温热部分,迅速浸泡在含有Coplin罐的二甲苯中。
  11. 在二甲苯中脱蜡两次,每次30分钟。确保这些部分几乎是半透明的。
  12. 通过甲醇或乙醇梯度系列(100%,90%,70%,50%,30%,D / W各5分钟)浸玻片。
  13. 用苏木精染色核(参见食谱)5-10分钟。

  14. 在流动的自来水中冲洗玻片2分钟
  15. 将载玻片放在含有Coplin罐的Picrosirius红色(见食谱)中1小时。
  16. 用新鲜的酸化水清洗滑梯两次(见食谱)(每次5分钟)。
  17. 用D / W清洗载玻片,通过甲醇或乙醇梯度(50%,70%,90%,每次5分钟)使切片脱水。
  18. 给100%乙醇(每个5分钟)三个变化。
  19. 在二甲苯中孵育30分钟(两次更换)。
  20. 挂在D.P.X.让它过夜。
  21. 观察明场或偏光显微镜下。
    使用常规数码相机或CCD拍摄图像

数据分析

  1. 苏木素染色细胞核紫罗兰色。然而,细胞核可能会出现黑色或棕色或灰色。在亮视野显微镜下,天狼星红染料染色后,胶原蛋白在浅黄色的细胞质背景上呈现粉红色至红色。 Picrosirius红色染色不仅提供关于组织样品中胶原蛋白分布的信息,而且还检测组织切片中的胶原纤维束。如果正在研究胶原纤维的破坏导致纤维束形成的中断,用这种方法对胶原蛋白染色可能是有价值的。
  2. 使用Picrosirius红色染色,我们可以证明在小鼠生殖组织中胶原的不同分布(图1)。为了证明该技术如何用于检测胶原蛋白降解,将小鼠绒毛膜蜕膜/羊膜用来自具有胶原酶活性的B群链球菌(GBS)的膜囊泡(MV)处理(Surve等,2016)。在正常组织中,在绒毛膜和羊膜中可见胶原束。然而,这在用GBS MV治疗时是分散的。在GBS MV处理的组织中清晰可见(图2)。


    图1. Picrosirius红色染料在各种小鼠生殖组织和胎膜中的胶原染色。红到粉红色是胶原蛋白染色。黄色是细胞质。比例尺= 50微米。


    图2.小鼠羊膜(A)和绒毛膜(C)中响应于来自GBS的膜囊泡(MV)的胶原降解。体内数据来自小鼠注射GBS MV在E16.5的羊膜囊中。离体绒毛羊膜与GBS MV孵育16小时。箭头显示胶原蛋白降解。比例尺= 50微米。

  3. 我们利用不同形式的胶原的双折射性质,通过偏振显微镜来证明它们在各种小鼠生殖组织中的分布(图3)。在偏光显微镜下,胶原蛋白I呈黄 - 红色(白色箭头),胶原蛋白III呈绿色(蓝色箭头),胶原蛋白IV呈弱至无双折射(白色轮廓的黑色箭头)(Montes and Junqueira,1991)。表1显示了这些发现的总结。


    图3.通过Picrosirius红色染料染色的小鼠组织的偏振显微镜检测胶原亚型。胶原蛋白I为黄红色(白色箭头),胶原蛋白III为绿色(蓝色箭头),胶原蛋白IV弱至无双折射(白色轮廓的黑色箭头)。比例尺= 50微米。

    表1.小鼠生殖组织和妊娠中胶原蛋白的分布,如Picrosirius红色染料染色和偏光显微镜检测

笔记

  1. 胶原纤维的染色及其分布在日常基础上是相当恒定的。
    使用相同的协议,染色可以用不同的组织重现
  2. 即使组织在较短时间内看起来染色,也不应在1小时之前取出。
  3. 在新鲜组织切片中观察到与在室温下储存超过2-3个月的那些相比,胶原染色没有差异。
    部分染色较早(2个月)与Picrosirirus红色显示相似的颜色和强度
  4. 双折射没有改变存储染色的部分。新鲜切片的双折射强度与在室温下染色和储存超过2-3个月的强度相似。

食谱

  1. 维格特的苏木精溶液
    1. 准备苏木素储备液
      1.0克苏木素
      100.0毫升96%乙醇
      正确混合并保持混合物1周,使其在室温下熟化
    2. 准备氯化铁(III)原液
      1.16克氯化铁(III)
      1.0毫升HCl(25%)
      99.0毫升蒸馏水
      充分溶解
    3. 将苏木精储备溶液与氯化铁(III)储液按1:1的比例混合,得到Weigert's苏木精染料溶液。使用前彻底混合
  2. 10%中性福尔马林缓冲液 0.4克NaH 2 PO 4 4 0.65克Na 2 HPO 4 4 90毫升蒸馏水
    10毫升甲醛
  3. 4%多聚甲醛
    4克多聚甲醛
    100毫升1x PBS
    65°C加热5分钟直至PFA溶解
  4. PBS的组成 0.8克NaCl
    0.144克Na 2 HPO 4 4 200毫克氯化钾
    240mg KH 2 PO 4 4 加100毫升D / W
  5. 聚赖氨酸涂层载玻片
    聚赖氨酸:10微升/盖玻片

    均匀地涂在干净的载玻片上表面 使其在室温下风干,并将其储存在室温下供进一步使用。没有必要洗
  6. Picrosirius红色解决方案
    0.1克直接红色80
    饱和苦味酸水溶液(1.3克,在100毫升蒸馏水中)
    轻轻搅拌至完全溶解
  7. 酸化水
    1毫升冰醋酸200毫升蒸馏水

致谢

MV Surve和S Bhutda承认UGC,Govt的奖学金。印度,阿尼尔也承认从CSIR,Govt。的印度。 N Singh承认政府生物技术部的初级研究奖学金。的印度。印度医学研究理事会(ICMR)和印度孟买种子基金会分别向D Modi和A Banerjee提供了财政援助。作者没有利益冲突。

参考

  1. Fujimoto,T.,Parry,S.,Urbanek,M.,Sammel,M.,Macones,G.,Kuivaniemi,H.,Romero,R.和Strauss,J.F.,3rd(2002)。基质金属蛋白酶-1(MMP-1)启动子中的单核苷酸多态性影响羊膜细胞MMP- 1表达和早产胎膜早破的风险。“生物化学杂志”277(8):6296-6302。
  2. Hampson,V.,Liu,D.,Billett,E。和Kirk,S。(1997)。 妊娠早产胎膜早破孕妇羊膜胶原含量及分布类型> Br J Obstet Gynaecol 104(9):1087-91。
  3. Junqueira,L. C.,Bignolas,G.和Brentani,R. R.(1979)。 Picrosirius染色加偏振显微镜,组织切片中检测胶原蛋白的具体方法 < em Histochem J 11(4):447-455。
  4. Lattouf,R.,Younes,R.,Lutomski,D.,Naaman,N.,Godeau,G.,Senni,K.和Changotade,S.(2014)。 Picrosirius红色染色:评估正常和病理组织中胶原网络的有用工具。
    J Histochem Cytochem 62(10):751-758。
  5. Marpaung,J。(2016)。 协会羊膜中胶原蛋白的厚度与发病率胎膜早破(5)(2):296-299。
  6. Montes G. S.和Junqueira L. C. U.(1991)。 使用picrosirius极化方法研究胶原蛋白的生物病理学。 Mem Inst Oswaldo Cruz 86(III):1-11。
  7. Sebire,N.J。(2001)。 Choriodecidual炎症综合征(CoDIS)是早期早产的主要原因,也是未被公认的原因,第二孕期流产。 Med假设 56(4):497-500。
  8. Shimizu,K.和Hokano,M.(1990)。 失去机械扩张对小鼠子宫中胶原蛋白降解的影响 J Anatomy 173:161-167。
  9. Surve,MV,Anil,A.,Kamath,KG,Bhutda,S.,Sthanam,LK,Pradhan,A.,Srivastava,R.,Basu,B.,Dutta,S.,Sen,S.,Modi,D 。和Banerjee,A。(2016)。 乙组链球菌膜泡破坏导致早产的胎儿 - 母体屏障出生。 PLoS Pathog 12(9):e1005816。
  10. VegaSánchez,R.,Estrada Gutierrez,G.,Cerbulo Vazquez,A.,Beltran Montoya,J.和Vadillo Ortega,F。(2004)。 胎盘绒毛间隙的特征是富含效应分子的环境,在分娩过程中诱发胎膜破裂。 / a> Ginecol Obstet Mex 72:593-601。
  11. Wang,H.,Parry,S.,Macones,G.,Sammel,MD,Ferrand,PE,Kuivaniemi,H.,Tromp,G.,Halder,I.,Shriver,MD,Romero,R. and Strauss,JF 3 RD 。 (2004年)。 功能上显着的SNP MMP8启动子单倍型和早产胎膜早破(PPROM)。


    Hum Mol Genet 13(21):2659-2669。

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Bhutda, S., Surve, M. V., Anil, A., Kamath, K. G., Singh, N., Modi, D. and Banerjee, A. (2017). Histochemical Staining of Collagen and Identification of Its Subtypes by Picrosirius Red Dye in Mouse Reproductive Tissues. Bio-protocol 7(21): e2592. DOI: 10.21769/BioProtoc.2592.
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