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The Assessment of Beta Cell Mass during Gestational Life in the Mouse

小鼠妊娠期 β 细胞质量的评估

YK Yury Kryvalap
JC Jan Czyzyk

发布: 2023年03月20日第13卷第6期 DOI: 10.21769/BioProtoc.4641 浏览次数: 1323

评审: RAMESH KUDIRAAnonymous reviewer(s)

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Abstract

Successful advancement in the treatment of diabetes mellitus is not possible without well-established methodology for beta cell mass calculation. Here, we offer the protocol to assess beta cell mass during embryonic development in the mouse. The described protocol has detailed steps on how to process extremely small embryonic pancreatic tissue, cut it on the cryostat, and stain tissue slides for microscopic analysis. The method does not require usage of confocal microscopy and takes advantage of enhanced automated image analysis with proprietary as well as open-source software packages.

Keywords: Beta cell mass (β细胞质量) search Insulin area (胰岛素区) search Embryonic pancreas (胚胎胰腺) search Timed pregnancy (定时妊娠) search Frozen sections (冰冻切片) search

Background

This protocol offers state-of-art detailed steps to assess beta cell mass on immunofluorescent (IF)-stained sections of pancreatic tissue. Quite frequently, beta cell area and mass estimation are heavily biased due to examination of too few (often just 2–3) sections of the pancreatic tissue. Here, we offer an unbiased novel approach to cut through the pancreatic tissue and make representative layers as well as backup slides for later staining. Using our approach, the number of sections and tissue levels that are necessary to accurately estimate the size of beta cell mass is dependent only on the pancreas developmental stage. Moreover, this protocol yields consistent estimations of beta cell mass among individual samples per single treatment group.

Materials and Reagents

  1. Pair of straight sharp non-serrated forceps (Fisher Scientific, catalog number: 12000127)

  2. Pair of 1 cc syringes (McKesson, catalog number: 16-ST1C) with 30.5 G needles (BD, catalog number: 305106)

  3. Petri dishes (Corning, catalog number: 351029)

  4. 500 mL Stericup Quick Release Millipore Express PLUS 0.22 μm filter unit (Millipore, catalog number: S2GPU05RE)

  5. 250 mL Stericup Quick Release Millipore Express PLUS 0.22 μm filter unit (Millipore, catalog number: S2GPU02RE)

  6. Super Pap-pen, small (Electron Microscopy Sciences, catalog number: 71312)

  7. Invisible tape (Staples, catalog number: 487908)

  8. Adhesive glass slide 75 × 25 × 1 mm (Matsunami, catalog number: SUMGP12)

  9. Immunofluorescence (IF) staining tray (Simport Scientific, catalog number: M9202)

  10. Neo Micro cover glass (Matsunami, catalog number: 24x50)

  11. Nail polish (Electron Microscopy Sciences, catalog number: 72180)

  12. Paper towel (Scott Kimberly-Clark Professional, catalog number: 09-24-589-0-06)

  13. 15 mL conical tubes (Thermo Fisher Scientific, catalog number: 339651)

  14. 50 mL conical tubes (Thermo Fisher Scientific, catalog number: 339653)

  15. Weigh boats (Electron Microscopy Sciences, catalog number: 70042)

  16. BALB/cJ male mice (The Jackson Laboratory, catalog number: 000651)

  17. Prechilled PBS pH 7.4 (Gibco, catalog number: 10010-023)

  18. Paraformaldehyde (PFA) 8% aqueous solution, EM grade (Electron Microscopy Sciences, catalog number: 157-8)

  19. Sucrose (Macron Fine Chemicals, catalog number: 8360-06)

  20. OCT compound (Scigen, catalog number: 4586)

  21. Biopsy cryomold (Sakura Tissue-Tek, catalog number: 4565)

  22. Dry ice pellets

  23. 100% ethanol

  24. Fisherfinest chemically resistant marker (Fisher Scientific, catalog number: 22-026-700)

  25. Bovine serum albumin (BSA) (fraction V) (Fisher Bioreagents, catalog number: BP1600-100)

  26. Sodium azide (Sigma, catalog number: S-8032)

  27. Anti-insulin mouse Alexa-647 (BD, catalog number: 565689)

  28. Anti-glucagon mouse Alexa-594 (SantaCruz, catalog number: sc-514592 AF594)

  29. Anti-somatostatin mouse Alexa-488 (BD, catalog number: 566032)

  30. Shandon Immu-Mount (Thermo Fisher Scientific, catalog number: 9990402)

  31. DAPI (4’,6-diamidino-2-phenylindole, dihydrochloride) (Thermo Fisher Scientific, catalog number: D1306)

  32. 30% sucrose (see Recipes)

  33. 5% BSA with 0.1% sodium azide (see Recipes)

  34. DAPI in slide mounting media (see Recipes)

Equipment

  1. Scissors (Fisher Scientific, catalog number: 13820002)

  2. Dissecting microscope with mirror and light below (Olympus, model: SZX7)

  3. Cryostat (Microm, model: HM 525)

  4. pH meter (Mettler Toledo, model: S220)

  5. IF-capable microscope or IF Virtual Slide scanner (Olympus VS 120, Zeiss AxioScan Z1)

  6. Analytical weigh scale (Mettler Toledo, model: XS105)

Software

  1. Visiopharm 2021.07 or later

    Alternatively:

  2. ImageJ with Trainable Weka Segmentation plugin (Arganda-Carreras et al., 2017).

Procedure

English
中文翻译

文章信息

版权信息

© 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).

如何引用

Kryvalap, Y. and Czyzyk, J. (2023). The Assessment of Beta Cell Mass during Gestational Life in the Mouse. Bio-protocol 13(6): e4641. DOI: 10.21769/BioProtoc.4641.

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分类

发育生物学 > 形态建成 > 细胞结构

细胞生物学 > 细胞成像 > 冷冻超薄切片

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