IMMUNOHISTOCHEMISTRY PROTOCOL WITH NOTES
Needed chemicals
• Ethanol
-> located in histology, CTM shelf (note: 100% EtOH is running low)
• Tissue Clear
-> located in histology, container beneath the sink
-> IMPORTANT: Tissue Clear is toxic and must be disposed of as chemical waste only
• ddH2O
-> located in genomic, the one from tap (milli-Q is not used)
• PBS
-> located in sorter room
• Goat Serum
-> stored in the freezer at -20°C in histology
• VECTOR Antigen Unmasking Solution (Citric Acid-based, pH 6,0); (H-3300)
-> stored at 4°C in histology
• Antibody-Diluent (Dako, Art.-Nr. S 302283-2)
-> stored at 4°C in histology
-> MBD has its own labeled bottle (opening date 26-01-2026)
• DAPI
-> stock located in protein lab, bottom shelf, left drawer
-> note: DAPI is more diluted for IHC usage compared with IF
• Mowiol
-> stored in sorter room, 4°C fridge
Needed Antibodies:
• Primaries:
-> TNNT2 (HPA015774) is in the red box inside one of the white boxes in the cardio freezer at -20°C
-> 4°C antibodies are now located in the sorter room
• Secondaries:
-> stored in the sorter room
-> use SAs raised in the same species as the blocking serum (if possible)
Needed Solutions:
Citrate-based Buffer solution pH 6.0
Add 2,5 ml Antigen Unmasking Solution (Citric Acid-based) to 250 ml ddH2O. Mix to dissolve.
Adjust pH to 8.0.
-> based on Rolfe’s protocol adjust to pH 8.0 (by adding NaOH)
-> since the pH meter’s response is slower, if the pH exceeds 8.0, add a small amount of HCl
-> as HCl is a strong acid, additional drops of NaOH will likely be required to return to pH 8.0
Antibody solutions
-> dilute all antibodies in Dako antibody diluent
-> note: Dako antibody diluent does the permeabilization - no additional permeabilization step needed
-> for one full slide approximately 600 µl of solution is needed
-> make the calculations in coherence with antibodies dilution rates (do not attempt to pipette less than 1 µl of antibody)
DAPI solution
Dilute 2.5 µl DAPI stock solution (10 mg/ml in ddH20) in 40 ml PBS
Useful numbers for calculations
-> Coplin jar volume: approximately 50 ml (for samples covering the whole slide, slightly greater volume is needed)
-> blue plastic container: 250 ml
-> slide: approximately 600 µl for all samples at one slide (but depends on how many samples are there, for chick samples 600 µl was sufficient)
WORKFLOW
DEPARAFFINIZATION and REHYDRATATION PROTOCOL
- Deparaffinize in Tissue Clear – 10 min
- Tissue Clear/100% Ethanol (1:1) – 5 min
- 100% Ethanol – 2 min
- 96% Ethanol – 2 min
- 70% Ethanol – 2 min
- 50% Ethanol – 2 min
- Aqua ddH20 – 2 min
-> more than 50 ml volume was required for the chick samples as they covered the entire slide
-> some prepared solutions are stored in the CTM cabinet next to the door in histology in a holder labeled
“MBD Katerina J”
-> PBS washing step between EtOH and ddH20 was omitted
-> if the solution volume does not fully cover the sample in the Coplin jar, additional ethanol may be added
-> exact ethanol concentrations are not critical; the key requirement is a decreasing ethanol gradient
-> EtOH is diluted in ddH20
CITRATE BASED BUFFER ANTIGEN RETRIEVAL PROTOCOL
- Pre-heat pressure cooker with water and dish containing Citrate Based Buffer until temperature reaches 90°C
- Immerse slides in staining dishes, close a cooker lid and set up indicator for cooking
without pressure! incubate 20 minutes at 90C
-> cooking program: Cake (used for both pre-heating and HIER)
-> lid-opening instructions are located on top of the cooker; open carefully
-> slides must be placed in the blue plastic container (do not use glass Coplin jars, as they may crack above ~70°C). - Switch off cooker, remove the staining dish to room temperature and allow the slides
to cool for 20 min.
-> remove the blue plastic container from the cooker - Wash sections in PBS 2 x 3 min
-> done in the Coplin jar
STAINING PROTOCOL
- Encircle sections with grease pen and sort slides into a damp chamber.
Block 60 min with 5% Goat Serum at r.t. (humidified chamber)
-> humidified chamber consists of three parts: box, lid, and slide holder
-> dampened tissues are used to prevent samples from drying out - Wipe away blocking buffer and incubate with primary antibodies overnight at 4°C (humidified chamber)
- Wash 3 x 5 min in PBS (cuvette)
- Incubate with secondary Abs 60 min (humidified chamber)
-> when performing multiple stainings on a single slide, do not mix secondary antibodies; use the same secondaries across the entire slide.
-> we’ve agreed on using 555 anti-mouse and 647 anti-rabbit (488 is not used because of the autofluorescence coming from the sample)
-> (alpha Sarc Act A7811 is mouse) - Wash 2 x 5 min in PBS (cuvette)
- Stain 20 min with DAPI solution at r.t. (cuvette, light protected)
- Wash 2 x 5 min in ddH2O (cuvette, light protected)
- Cover slip and embed in Mowiol (briefcase)
-> if the cover slip is not clear wash it before use, then spray it with ethanol to speed up the drying process, and wipe it with a tissue
-> the ones in histology are perfectly fine
Postprocessing
- Let the sample in the fridge over night.
- In the morning use nail polish to fix the cover slip
-> IMPORTANT: Use non-fluorescent nail polish. The one used in histology (black lid) is suitable. - Scan the whole slide (before bleaching it at confocal)
- Do the imaging using confocal / …