IHC preparation

An overview of the reagents and equipment required for an IHC experiment.

Immunohistochemistry Materials And Equipment

• Frozen or paraffin-embedded tissue
• Cryo-embedded media (e.g., OCT) 
• Sucrose 
• 4% Paraformaldehyde (PFA) 
• Microtome 
• Glass slides 
• Coverslips 
• Refrigerator 
• Incubator 
• Xylene 
• Ethanol (different percentages)
• Boiling source 
• Primary and secondary antibodies 
• Hematoxylin 
• Serum 
• Citrate buffer (pH 6) 
• Tris-EDTA buffer (pH 9) 
• Diaminobenzidine tetrachloride (DAB) 
• Microscope

Solutions

Citrate Buffer	For 1000 ml	1x TBS	For 1000 ml
10 mM Trisodium citrate ₂H₂O	2.9 g	20 mM Tris-base	2.4 g
1.9 mM citric acid H₂O	0.4 g	150 mM NaCl	8.7 g
Adjust pH to 6.0		Adjust pH to 7.6
Add ddH₂O to 1000 ml		Add ddH₂O to 1000 ml

Tissue slide handling

Tissue sections should be placed on a glass slide. Allow adhesion of tissue to slide by air drying and additional baking in an incubator. Cut and unbaked slides can be stored at 4°C for later use. However, storage might have an effect on the antigenic potential and this varies for each protein. Therefore, it is recommended to minimize storage time or to use freshly prepared slides.