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.
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.
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.
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.
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.
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.
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
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.
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.
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.