Immunofluorescence Protocol for Frozen Tissues with Citrate Retrieval

A. Solutions and Reagents

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

1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS, add 100 mL 10X Wash Buffer, Phosphate Buffered Saline (PBS) #12528 to 900 mL water, mix. Adjust pH to 8.0.
2. 16% Formaldehyde, Methanol-Free #12606: Use fresh. Dilute to 4% in 1X PBS prior to 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. Store at 4°C.
6. Fluorochrome-conjugated Secondary Antibody: Use a secondary antibody that is reactive to your primary antibody host species (e.g., rabbit). Click here for a list of secondary antibodies approved for immunofluorescence.
7. Mounting Medium: Use Prolong Gold AntiFade Reagent #9071 or Prolong Gold AntiFade Reagent with DAPI #8961.

B. Tissue Preparation and Antigen Retrieval

1. Fixation and Cryosectioning: Preserve tissue antigens by fixing with a high-quality formaldehyde diluted to 4% in 1X PBS. Use fresh and discard unused fixative appropriately. For best results, perfuse animals according to approved protocols.
   1. For tissue stored at -80°C, remove from freezer and equilibrate to your sectioning temperature for approximately 15 min before attempting to section. This may prevent cracking of the block when sectioning.
   2. Section tissue at a range of 6-20 µm and adhere to positively charged slides.
   3. For fresh-frozen samples, fix for 15 min at room temperature. Rinse slides briefly with 1X PBS.
2. Antigen Retrieval: Immerse slides in 1X citrate unmasking solution, heat in a microwave until boiling begins, and maintain a temperature of approximately 70°C for 20 min. Cool slides on the benchtop for 30 min.

C. Immunostaining

NOTE: All subsequent incubations should be performed in a humid chamber at room temperature, unless otherwise noted. Do not allow samples to dry at any time during this procedure, and protect sensitive fluorophores from light.

1. Wash three times with 1X PBS for 5 min each.
2. Block specimen in Blocking Buffer for 60 min.
3. While blocking, prepare each primary antibody by diluting it in Antibody Dilution Buffer as indicated on the product webpage or datasheet.
4. Aspirate blocking solution, then apply diluted primary antibody.
5. Incubate overnight at 4°C.
6. Wash three times with 1X PBS for 5 min each.
7. Incubate samples with fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1-2 hr, protected from light.
8. Wash three times with 1X PBS for 5 min each.
9. Counterstain as appropriate, then mount samples for imaging.
10. For long-term storage, store stained samples at 4°C, protected from light.

For Research Use Only. Not for Use in Diagnostic Procedures.