Here are some informations and advice to guide you in the design of your immunostainings.
Tutorials et useful documents
- Tissue Controls & More for Immunohistochemistry (CSTTechTips)
- How to perform Antigen retrieval (AR) in Immunohistochemistry (IHC) (CSTTechTips)?
- How does antibody diluent affect staining in Immunohistochemistry (IHC) (CSTTechTips)?
- 5 Tips to Start Your Multiplex IHC Optimization (CSTTechTips)
- Multiplex IHC Optimization: Antibody Titration & Fluorophore Pairing (CSTTechTips)
- IHC world FAQ
- Optimisation IHC/ICC (webinar Abcam)
- IHC/ICC simple ou multiples marqueurs (webinar Abcam)
Protocol optimization advice
- Check the recommendations indicated on the datasheet and in the literature
- Check the solutions (pH, precipitation, age, ...). Antibodies do not like crystallization -> dilute them in 50% sterile glycerol for storage at -20 ° C
- Start with a standard protocol on + and - controls with 3 dilutions of Ac (between 1 and 10 µg/ml)
- Do not hesitate to rinse your blades well !!
- Troubleshootong tips (SABbiotech)
Which controls ?
Positive controls
Which one ?
- Tissue, cells, (cell pellets) (préparés de la même façon vos échantillons)
- Other primary antibody
- Other detection method
What to check ?
- Localization of the labeling (cell type, subcellular compartment)
- Use, if necessary, co-labeling with organelle markers or cell-type specific markers
Negative controls
Specific binding of primary Ab ?
- +++ mouse / KO +++ cells (check epitopes if they are truncated proteins)
- Blocking peptide
Specific binding of revelation reagents (secondary Ab, substrate) ?
- Omission of primary Antibody
- Isotype control
- For multiplex: each primary Ab + all secondary Ab (use cross-adsorbed secondary Ab !!)