2.13. Immunofluorescence Staining of the Kidney Sections

Immunofluorescence Staining of the Kidney Sections

Deparaffinization:

Place the slides in oven for 1 h at 65 °C

Place the slides in a rack, and perform the following washes: Xylene: 2 x 5 minutes

100% ethanol: 2 x 5 minutes

95% ethanol: 2 x 5 minutes

70 % ethanol: 2 x 5 minutes

ddH₂O: 2 x 5 minutes

Antigen retrieval and Immunofluorescence staining

• For antigen retrieval set up the vegetable steamer and preheat. Pre-heat the antigen retrieval buffer (10 mM citric acid) to boiling in a flask (a microwave is handy for this). Put the slides in the pre-heated buffer, keep the buffer with the slides in the steamer for 25 minutes.
• After the antigenretrieval step wash the slides3 x 5 minutes in TBS plus 0.1% Tween^® 20 detergent with gentle agitation. Block in 10% goat serum in PBS for 1 h at room temperature. Drain slides for a few seconds (do not rinse) and wipe around the sections with tissue paper.

Apply the followingprimary antibody diluted(as indicated below) in TBS with 10% goat serum.

• Anti-PPIA- Polyclonal Antibody produced in Rabbit, Catalog # PA1-025 (Invitrogen) dilution 1/200 incubation overnight at room temperature.
• Anit-Fn1 antibody Polyclonal Antibody produced in Rabbit, Catalog # F3648-100UL (Sigma) dilution 1/400, incubation overnight at room temperature.
• Anti-Collagen I antibody, Mouse monoclonal Catalog # MA1-26771 (Invitrogen), dilution 1/500, incubation overnight at room temperature.

Rinse 3 x 5min TBS plus 0.1% Tween^® 20 detergent with gentle agitation.

Apply fluorophore-conjugated secondary antibody: fluorescence Alexa 555–conjugated goat anti-rabbit (1/1000) or Alexa 488–conjugated goat anti-mouse secondary antibodies (1/500) (Invitrogen) to the slide and incubate for 1 h at room temperature. Rinse 3 x 5min TBS plus 0.1% Tween^® 20 detergent with gentle agitation. Mount with Vectashield 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories) to visualize the nuclei.