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Immunohistochemistry Protocol for Frozen TissueDOWNLOAD A PDF
This protocol is a recommendation only. Please optimize the procedure since experimental conditions can vary for different tissue samples.
Tissue PreparationNote: For tissue stored at -80°C, remove from freezer and equilibrate at -20°C for about 15 minutes before sectioning. This prevents cracking of the block during sectioning.
- Cut sections at 4-8 μm and place on pre-cleaned and positively charged microscope slides.
- Air dry sections on bench for a few minutes to help the sections adhere to the slides.
- Fix sections in precooled acetone for 10 minutes at 4°C.
- Wash with Tris-Buffered Saline (TBS) to remove all traces of acetone.
Recommended: Do not allow tissues to dry at any time during the staining procedure.
- Rinse slides 2 times with Tris-Buffered Saline + Tween (TBST) for 5 minutes each at RT.
- Block with 5% serum or BSA for 2 hours at RT.
- Drain blocking buffer from slide.
- Incubate slides with the diluted primary antibody overnight at 4°C with gentle agitation.
- Wash slides 2 times with TBST for 5 minutes at RT.
- Incubate slides with diluted conjugated secondary antibody for 2 hours at RT with gentle agitation.
- Wash slides 2 times with TBST for 5 minutes at RT.
- Develop with chromogen for 10 minutes at RT.
- Wash slides in distilled water for 1 minute at RT.
- Counterstain (if required).
- Mount coverslips.
Immunohistochemistry Protocol for Paraffin Embedded Tissue DOWNLOAD A PDF
This protocol is a recommendation only. Please optimize the procedure since experimental conditions can vary for different tissue samples.
Tissue Preparation (Formalin Fixed, Paraffin-embedded Sections)- Cut sections at 4um and place on pre-cleaned and positively charged microscope slides.
- Heat in a tissue-drying oven for 45 minutes at 60°C.
- Wash slides 2 times in Xylene for 3 minutes each time at room temperature (RT).
- Wash slides in Xylene 1:1 with 100% ethanol for 3 minutes at RT.
- Wash slides 2 times in 100% ethanol for 3 minutes each at RT.
- Wash slides 2 times in 95% ethanol for 3 minutes each at RT.
- Wash slides in 70% ethanol for 3 minutes at RT.
- Wash slides in 50% ethanol for 3 minutes at RT.
- Rinse slides gently with running distilled water for 5 minutes at RT.
- Boil slides in 0.01M sodium citrate buffer (pH6) at 100°C for 15-20 minutes. Remove the slides from heat and allow them to stand at RT in buffer for 20 minutes.
- Rinse twice with Tris-Buffered Saline + Tween (TBST) for 5 minutes at RT.
Recommended: Do not allow tissues to dry at any time during the staining procedure.
- Block endogenous peroxidase with 3% hydrogen peroxide for 30 minutes.
- Block with 5% serum or BSA for 2 hours at RT.
- Drain blocking buffer from slide.
- Incubate slides with the diluted primary antibody overnight at 4°C with gentle agitation.
- Wash slides 2 times with TBST for 5 minutes at RT.
- Develop with chromogen for 10 minutes at RT.
- Wash slides in distilled water for 1 minute at RT.
- Counterstain (if required).
- Dehydrate when using a chromogen substrate that is alcohol insoluble by washing slides in 80%, 95%, 100% and Xylene each for 1 minute at RT.
- Mount coverslips.