Protocol & Procedures Document 

Protocol Name 

Frozen section staining for immunofluorescence (IF) microscopy 

Including tyramide signal amplification (TSA) 

Materials Required 

  • Prepare TBS-T Wash Buffer 

    • 0.1 M TRIS-HCl, pH 7.5 (100 ml 1M Tris/HCl, pH 7.5) 

    • 0.15 M NaCl (8.8g NaCl) 

    • 0.001-0.1% Tween®20 (I usually use 1mL in 1L) 

    • MilliQ water up to 1L 

  • Cut OCT-embedded tissue (7 μm preferably) 

  • Wet chamber to provide humidity to slides during incubation 

  • Coplin jar 

  • Blocking buffer (casein, 0.5% BSA/PBS, serum or equivalent) 

  • Primary antibodies &/or fluorescently labelled secondary antibodies &/or Fluorescent markers (e.g. Phalloidin, Cell mask, etc.) 

  • DAPI 

  • Mounting media (IMBiol, Prolong Diamond, etc.) 

  • #1.5 coverslips 

  • Sealant (clear nail polish) 


  1. Dry the sections o/n (or at least 1 hour) at room temperature.  

  2. Put the slides into the Coplin jar for washes 

  3. Wash 3-6 x 3 minutes in TBS-T (to dissolve the OCT) 

  4. Draw circles around the tissue with a hydrophobic pen or just dry the slide around the tissue before each incubation step to provide surface tension 

  5. If your tissue was fresh frozen, fix the tissue for 10 minutes in 75% acetone + 25% ethanol or 4% PFA -> Wash 3-6 x 3 minutes in TBS-T 

  6. If using tyramide signal amplification (TSA): Add approx. 100 μl 1 % peroxide (dilute in MQ water) and wait until you see bubbles (3 minutes) -> Wash 3 x 3 minutes in TBS-T 

  7. If using primary labelled or primary followed by secondary antibody, skip peroxide step. 

  8. Block non-specific binding, generally related to isotype. General block is casein/Background sniper (for specific antibodies, the protein found in milk powder). Can use undiluted or anywhere from 1:10 to 1:100 depending on your antibody 

  9. Dilute primary antibody in appropriate diluent  (pH of diluent is antibody specific, read the datasheet: primary antibody dilution is determined empirically through testing and is different for every antibody: use approx. 200 μl / sample) 

  10. Incubate for 30-120 minutes in the wet chamber at room temp, overnight at 4 degrees 

  11. Wash 6 x 3 minutes in TBS-T 

  12. If using primary fluorescently labelled antibody, skip to 15 

  13. if using secondary fluorescently conjugated antibody, use 1:500 dilution of secondary antibody  

  14. If using TSA,  detect the primary antibody by using the correct Anti-XXX IgG 

    • ImmPRESS HRP Anti-XXX IgG (Peroxidase) Polymer Detection Kit, Vector Laboratories: 

      • Dilute 1:10 in TBS-T 

      • Incubate for 15 minutes in the wet chamber 

    • Wash 6 x 3 minutes in TBS-T 

    • TSA flurophore of interest (Perkin Elmer): 

      • Dilute 1:50 in the provided diluent (use approx. 100 μl / sample; you can increase the dilution even further, but the diluent is the limiting factor) 

      • Incubate for 7-10 minutes in the wet chamber (covered!) 

  15. Wash 6 x 3 minutes in TBS-T 

  16. Add DAPI 

    • Dilute 1 : 20-35,000 in PBS 

    • Incubate for 10 minutes in the wet chamber  

  17. Wash 2 x 3 minutes in TBS-T 

  18. Wash 1 x in MQ water to remove debris from slide, dry around tissue. 

  19. Wash coverslip once with MQ water to remove debris from coverslip. 

  20. Add a drop of mounting media onto the tissue. Place dry coverslip onto mounting media. Do not create bubbles between the slide and coverslip, if bubbles occur, push them out the side.  

  21. Seal the slide with a sealant or let the mounting media cure for at least 60 minutes. 

  22. Cleaning slide: Always clean both slide and coverslip. 

  23. The best images are collected directly after staining. But slides stained like this can be stored short-term at 4 °C, or frozen at -20 °C and stored for later analysis. 


  • Labels such as Alexa dyes, JFs, or SiR will last longer than expressed protein labels such as GFP/mCherry 

  • Number 1.5 coverslips are best and are available in the IMB store (Cat# D766) 


V1 – Mar 2020