- In the frozen tissue IHC protocol at https://www.rndsystems.com/resources/protocols/protocol-preparation-and-fluorescent-ihc-staining-frozen-tissue-sections, how is isopnetane mixed with dry ice?
- In this protocol, isopentane and dry ice are mixed together in a container.
- What coverslips and cell culture plates are recommended for ICC stainings on coverslips?
- In house, we use 12 mm Cover Glasses from Carolina, Catalog # 633009. We use 24-well flat bottom tissue culture treated cell culture plates from Corning, Catalog # 3526.
- What is the concentration of the biotinylated secondary antibody in the Cell & Tissue Staining Kit?
- The concentraion of the secondary antibody is considered proprietary. The protocol instructs adding 1-3 drops of the secondary antibody directly to the tissue section.
Cell and Tissue Staining Kits
- Are deparaffinization and antigen retrieval performed prior to starting IHC detection using Cell and Tissue Staining Kits (for example, Catalog # CTS019)?
- Yes, perform deparaffinization and, if necesary, antigen retrieval prior to treating samples with primary antibody, and then use the Cell and Tissue Staining Kit for detection.
Immunohistochemistry Reagents
- Are the Hydrophobic Coverslips, Catalog # 4867-100, reusable?
- We do not recommend reusing the coverslips.
- Can an R&D Systems antibody which has been validated for Immunocytochemistry (ICC or IF) be used in Immunohistochemistry (IHC)? Can an antibody which has been validated for IHC be used in ICC?
- R&D Systems will support an antibody that has been validated in-house for ICC or IHC in both applications. Although we cannot guarantee that an antibody will work in all cells or tissues, under all conditions, our validation shows that the antibody recognizes the fixed antigen. We may not have tested the antibody in both ICC and IHC, but R&D Systems will support its use in either application.
- Do you have any tips to reduce autofluorescence when staining cells using Flow Cytometry, ICC or IHC?
- There are products in the market available to reduce autofluorescence. Alternatively, the fluorophore can be changed to one that falls in the red or far red region, as there is less autofluorescence in that region. Unstained samples can also be checked to see which channel produces the least autofluorescence. Lasltly, one can use chromogenic detection methods if autofluorescence is difficult to eliminate.
- For Heat Induced Epitope Retrieval of paraffin-embedded tissue samples, what should be the pH of the retrieval buffer?
- In house, we most commonly use basic antigen retrieval buffer at pH 9 (Catalog # VCTS021). However, antigen retrieval depends on the tissue and one should always test if antigen retrieval is necessary and also test other antigen retrieval conditions to determine what works best for the specific tissue being stained. R&D Systems offers ready-to-use Antigen Retrieval reagents at various pHs - Catalogs #s VCTS021, VCTS022, and VCTS023.
- How many Hydrophobic Coverslips are provided in each package of Catalog # 4867-100?
- Each package contains 100 coverslips.
- What blocking serum is recommended for IHC/ICC?
- A serum identical to the host animal of the secondary antibody or serum from an unrelated species is recommended for blocking. In-house, we regularly use donkey serum for blocking. See our IHC/ICC protocols on our website:https://www.rndsystems.com/resources/protocols/immunohistochemistry-immunocytochemistry-protocols
- What coverslips and cell culture plates are recommended for ICC stainings on coverslips?
- In house, we use 12 mm Cover Glasses from Carolina, Catalog # 633009. We use 24-well flat bottom tissue culture treated cell culture plates from Corning, Catalog # 3526.
- What is the difference between fixation for ICC using methanol and acetone versus using 4% PFA?
- Acetone, ethanol, and methanol are precipitating fixatives. They precipitate proteins at their isoelectric point, whereas formaldehyde is a cross-linking fixative. Mixing both types of fixatives may allow for better fixation of proteins.
- What is the percent light transmission for the Hydrophobic Coverslips, Catalog # 4867-100?
- The coverslips transmit greater than 90% of visible light and are slightly opaque to UV light.
- What peroxidase blocking reagent should be used for frozen and paraffin-embedded sections?
- Our recommendation is to use 0.3% H2O2 in methanol. This is particularly beneficial for fragile tissues such as frozen samples. Methanol itself is a peroxidase inhibitor and mixing it with 0.3% H2O2 produces an additional quenching effect. 3% H2O2 in PBS may be too harsh on tissues.