TITLE: Immunofluorescent Staining of Mouse Pancreas for Islet Cell Mass Analysis
AUTHORS: Islet  and Pancreas Analysis Core

[DESCRIPTION]
This SOP defines the assay method used by the Vanderbilt Diabetes Center Islet and Pancreas Analysis (IPA) Core for quantitative determination of the islet cell composition and islet cell mass of mouse pancreas by immunofluorescent staining.

[STEPS]
SECTION: Immunofluorescent staining
7. Gather reagents for immunostaining, noting that steps 9-11, 15, and 17-18 can be performed in Kartell staining chambers (each holds ~50 mL).
 

SECTION: Imaging and analysis
21. Acquire images of stained pancreas sections using a high-resolution whole slide scanning system (Aperio ScanScope FL, Leica Biosystems). An example field of view is shown below in Figure 1.
SECTION: Immunofluorescent staining
8. Let the frozen cryosections thaw at room temperature and air-dry for about 30 min.


SECTION: Immunofluorescent staining
9. To remove OCT, wash slides 3 times in 1X PBS for 5 min, decanting or switching to a new chamber after each wash.
SECTION: Immunofluorescent staining
10. Permeabilize the tissue section with permeabilization solution for 15 min at room temperature.
SECTION: Immunofluorescent staining
11. Wash the sections 3 times in 1X PBS for 5 min, decanting or switching to a new chamber after each wash.
SECTION: Immunofluorescent staining
12. Draw rectangles around the sections with PAP pen and let them dry for about 5 min.
SECTION: Immunofluorescent staining
13. Block the sections with blocking buffer at room temperature for 90 min in a humidified chamber. Use a sufficient volume (approximately 200-300 µl) to ensure each section is entirely covered with buffer.
SECTION: Immunofluorescent staining
14. Aspirate the blocking buffer, add primary antibodies diluted in antibody buffer (see Table 1), and incubate in a humidified chamber   at 4 °C. Use a sufficient volume (approximately 200-300 µl) to ensure each section is entirely covered with antibody solution.

     
  
SECTION: Immunofluorescent staining
15. Aspirate the primary antibody solution and wash the sections 3 times in wash buffer for 10 min, decanting or switching to a new chamber after each wash.
SECTION: Immunofluorescent staining
16. Add secondary antibodies diluted in antibody buffer (see Table 1) and incubate for 90 min at room temperature in a humidified chamber. Use a sufficient volume (approximately 200-300 µl) to ensure each section is entirely covered with antibody solution.
SECTION: Immunofluorescent staining
17. Aspirate the secondary antibody solution and incubate slides in DAPI solution to counterstain for 10 min at room temperature.
SECTION: Immunofluorescent staining
18. Wash slides 3 times in 1x PBS for 15 min, decanting or switching to a new chamber after each wash.
SECTION: Immunofluorescent staining
19. Add a sufficient volume of SlowFade Gold mounting medium (approximately 5-10 µl) to each tissue section, ensuring tissue section will be entirely covered after medium spreads. Carefully mount coverslips, making sure to avoid creating air bubbles between the tissue and coverslip.
SECTION: Immunofluorescent staining
20. Blot away any excess mounting medium from slide edges and seal the coverslipped slides with nail polish on all edges. Allow slides to dry completely before imaging.
SECTION: Imaging and analysis
22. Apply a tissue classifier algorithm (HALO® image analysis platform, Indica Labs) to quantify cross-sectional area of islet alpha (glucagon+), beta (insulin+), and delta (somatostatin+) cells. An example markup image is shown in Figure 2.

   
 

SECTION: Reagent preparation
1. 10% Triton X-100 stock (30 mL) – Combine 3 mL Triton-X-100 and 27 mL 1X PBS. Mix on shaker for 30 min or until Triton X-100 is completely dissolved. Store at 4 °C for up to 1 month.
SECTION: Reagent preparation
2. Permeabilization solution (0.2% Triton, 50 mL) – Combine 1 mL 10% Triton stock and 49 mL 1X PBS.
SECTION: Reagent preparation
3. Blocking buffer (5% Normal Donkey Serum, 4 mL) – Combine 0.2 mL NDS and 3.8 mL 1X PBS.
SECTION: Reagent preparation
4. Antibody buffer (10 mL) – Combine 0.1 g BSA, 0.1 mL 10% Triton stock, and 9.8 mL 1X PBS. Filter solution through a 0.22 μm syringe filter and store at 4 °C.
SECTION: Reagent preparation
5. DAPI staining solution (1:25,000, 50 mL) – Combine 2 µl DAPI stock (5mg/mL) and 50 mL 1X PBS. Keep protected from light or prepare right before step 17.
SECTION: Reagent preparation
6. Wash buffer (150 mL) - Combine 148.5 mL 1X PBS and 1.5 mL 10% Triton X-100 stock.