Immunofluorescence (Frozen Tissues with Citrate Retrieval)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
2. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
3. 1X Citrate Unmasking Solution: To prepare 250 mL of 1X citrate unmasking solution, dilute 25 ml of SignalStain^® Citrate Unmasking Solution (10X) (#14746) with 225 mL of dH₂O.
4. Blocking Buffer: (1X PBS / 5% normal serum / 0.3% Triton^™ X-100): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425) to 9.5 ml 1X PBS) and mix well. While stirring, add 30 µl Triton^™ X-100.
5. Antibody Dilution Buffer: (1X PBS / 1% BSA / 0.3% Triton^™ X-100): To prepare 10 ml, add 30 µl Triton^™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (#9998), mix.
6. Recommended Fluorochrome-conjugated Anti-mouse secondary antibodies:
   • Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 488 Conjugate) #4412
   • Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 555 Conjugate) #4413
   • Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 594 Conjugate) #8889
   • Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 647 Conjugate) #4414
7. NOTE: When using any primary or fluorochrome-conjugated secondary antibody for the first time, titrate the antibody to determine which dilution allows for the strongest specific signal with the least background for your sample.
8. Prolong^® Gold AntiFade Reagent (#9071), Prolong^® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation and Antigen Retrieval

NOTE: Application of traditional antigen retrieval procedures to fixed-frozen tissues may result in excessive liberation of sections from the slide. It is highly recommended that users pre-treat glass slides with a reagent that promotes tissue adherence.(e.g., HistoGrip^™) while reducing retrieval temperature to 70°C.

1. For tissue stored at -80°C: remove from freezer and equilibrate at -20°C for approximately 15 minutes before attempting to section. This may prevent cracking of the block when sectioning.
2. Section tissue at a range of 6-20 µm and place on positively charged slides.
3. Bake on a slide warmer at 60°C for 1 hour (this helps sections adhere to slides).
4. Submerse slides in 1X citrate unmasking solution, heat in a microwave, and maintain a temperature of ~70°C for 20 minutes.
5. Cool slides on bench top for 30 min.
6. Rinse sections briefly in 1X PBS.

Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

1. Block specimen in Blocking Buffer for 60 min.
2. While blocking, prepare primary antibody by diluting as indicated on protocol on product webpage in Antibody Dilution Buffer.
3. Aspirate blocking solution, apply diluted primary antibody.
4. Incubate overnight at 4°C.
5. Rinse three times in 1X PBS for 5 min each.
6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1-2 hr at room temperature in the dark.
7. Rinse three times in 1X PBS for 5 min each.
8. Coverslip slides with Prolong^® Gold Antifade Reagent (#9071) or Prolong^® Gold Antifade Reagent with DAPI (#8961).
9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.