Immunological analyses (IF on Paraffin Sections)

IF on Paraf*in Sections

SAMPLE COLLECTION

1. Dissect and perform quick washes of gonads with 1x PBS.

Perform steps 2 & 3 at 4°C with gentle agitation on an orbital shaker.

2. Fix with 4% PFA, overnight.

3. Wash gonads with 1x PBS for 10 mins. Repeat 3x.

4. Embed in parafIin using standard protocols. If needed, refer to Bilinska, Hejmej, & Kotula-Balak 2018, Preparation of Testicular Samples for Histology and Immunohistochemistry, section 3.1.1.

5. Section gonads at 5um and store at 4°C for approximately <1 year.

IF DAY 1

For deparifEinization, rehydration, and antigen retrieval, perform one of the following: Steps 6-7 or 8.

6. Allow slides to reach room temperature, deparafIinize and rehydrate sections: 10 minutes in xylene, repeat 2x; then 5 minutes each in decreasing concentrations of ethanol: 100%, 95%, 70%, 40%. Place slides in sterile water.

7. Perform heat-induced antigen retrieval using a pressure cooker with in 10 mM sodium citrate buffer pH6, 0.1% Tween-20, made fresh.

8. Use PT Link machine with pH6 buffer.

9. Allow slides to return to room temperature, create boundary around sections using a hydrophobic barrier pen, and block with 10% normal donkey serum (NDS), 3% bovine serum albumin (BSA), 0.1% Tween-20 made in PBS for 1 hour, room temperature, in a humidiIied chamber.

10. Incubate with primary antibody in 3% NDS, 3% BSA, 0.1% Tween-20 in PBS, overnight at 4°C, in humidiIied chamber. All antibodies (manufacture, dilution used) used are listed in the Key Resources Table.

IF DAY 2

Perform steps 11 & 13 at room temperature, in the dark, with gentle agitation on an orbital shaker.

11. Wash slides in 0.1% Tween-20 made in 1x PBS for 10 minutes. Repeat 3x.

12. Incubate with secondary antibody for 1 hour in 0.1% Tween-20 made in 1x PBS at room temperature, in dark, and in a humidiIied chamber. For SOX9 and DMRT1 we used Alexa Flour 555 diluted at 1/200.

14. Wash slides in 1x PBS for 10 minutes. Repeat 3x.

15. Mount slides with Vectashield containing DAPI.

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