Immunofluorescence Protocol for Frozen Tissue (IF-F)

IMPORTANT: Please refer to the Product Usage Information section on the product webpage or datasheet to determine if your product is validated and approved for use on frozen tissue sections (IF-F).

A. Solutions and Reagents

Achieve higher quality immunofluorescent images using the efficient and cost-effective, pre-made reagents in our Immunofluorescence Application Solutions Kit #12727.

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

1. 1X Phosphate Buffered Saline (PBS): To prepare 1L of 1X PBS, add 100 mL 10X Wash Buffer, Phosphate Buffered Saline #12528 to 900 mL dH2O, mix. Adjust pH to 8.0.
2. 4% Formaldehyde, Methanol-Free #47746: Use fresh.
3. Blocking Buffer: Purchase ready-to-use Immunofluorescence Blocking Buffer #12411, or prepare a 1X PBS / 5% normal serum / 0.3% Triton X-100 buffer by adding 0.5 mL normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum #5425) and 30 µL Triton X-100 to 9.5 mL of 1X PBS. Store at 4°C.
4. Antibody Dilution Buffer: Purchase ready-to-use Immunofluorescence Antibody Dilution Buffer #12378, or prepare a 1X PBS / 1% BSA / 0.3% Triton X-100 buffer by adding 0.1 g BSA #9998 and 30 µL Triton X-100 to 10 mL of 1X PBS. Store at 4°C.
5. 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.
6. Mounting Medium: Use Prolong Gold AntiFade Reagent #9071 or Prolong Gold AntiFade Reagent with DAPI #8961.
7. Optional Solutions

IMPORTANT: Some CST^® antibodies may require optional preparation steps for optimal results. Please refer to the protocol on the product webpage for detailed recommendations.
1. 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.
2. 100% Methanol #13604: Chill before use.

B. Tissue Preparation

1. Fixation: 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 fresh-frozen samples, section tissue at a range of 6-20 µm, adhere to positively charged slides, and then fix for 15 min at room temperature.
2. Methanol Permeabilization (optional): Cover tissue sections with ice-cold 100% methanol and incubate for 10 min at –20°C or on ice. Do not allow sections to dry.
3. Antigen Retrieval (optional): Submerse slides in 1X citrate unmasking solution, heat in a microwave until boiling is initiated, and maintain a temperature of ~70°C for 20 min. Cool slides on the benchtop for 30 min.

C. Immunostaining

NOTE: Do not allow samples to dry at any time during this procedure, and protect sensitive fluorophores from light.

1. Rinse slides briefly with 1X PBS.
2. Block specimen with 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 and then apply diluted primary antibody.
5. Incubate overnight at 4°C.
6. Wash three times with 1X PBS for 5 min each.

NOTE: If using a fluorochrome-conjugated primary antibody, then skip to Step 9.

7. Incubate specimen with fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hr protected from light.
8. Wash three times in 1X PBS for 5 min each.
9. Counterstain as appropriate and then mount samples for imaging.

Contact us with any questions.

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