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It is also possible to use commercially available universal blocking buffers. Other common blocking buffers include normal serum, non-fat dry milk, BSA, or gelatin. [5] [6] Endogenous enzyme activity may also cause background staining but can be reduced if the tissue is treated with hydrogen peroxide. [5]
One of the main difficulties with IHC staining is overcoming specific or non-specific background. Optimisation of fixation methods and times, pre-treatment with blocking agents, incubating antibodies with high salt, and optimising post-antibody wash buffers and wash times are all important for obtaining high quality immunostaining.
After blocking, a solution of primary antibody (generally between 0.5 and 5 micrograms/mL) diluted in either PBS or TBST wash buffer is incubated with the membrane under gentle agitation for typically an hour at room temperature, or overnight at 4°C. It can also be incubated at different temperatures, with lesser temperatures being associated ...
The newly transferred blots are then soaked in a blocking solution; non-fat milk and bovine serum albumin are common blocking buffers. [8] This blocking solution assists with preventing non-specific binding of the primary and/or secondary antibodies to the nitrocellulose membrane. Once the blocking solution has adequate contact time with the ...
The chemical composition and pH value of the buffer solution also contribute to the effectiveness of heat-induced antigen retrieval. [1] Thus, the AR-immunohistochemistry protocol must be optimized for each tissue type, fixation method, and antigen using a "test battery" to maximize antigen recovery in formalin fixed paraffin embedded sections.
3,3′,5,5′-Tetramethylbenzidine or TMB is a chromogenic substrate used in staining procedures in immunohistochemistry as well as being a visualising reagent used in enzyme-linked immunosorbent assays . [1] TMB is a white solid that forms a pale blue-green liquid in solution with ethyl acetate.
Immunohistochemistry with cytokeratin 5/6 antibodies in usual ductal hyperplasia, showing a mosaic pattern, predominantly in the central zone. Cytokeratin 5/6 antibodies are antibodies that target both cytokeratin 5 and cytokeratin 6. [1] These are used in immunohistochemistry, often called CK 5/6 staining, [2] including the following applications:
308 11747 Ensembl ENSG00000164111 ENSMUSG00000027712 UniProt P08758 P48036 RefSeq (mRNA) NM_001154 NM_009673 RefSeq (protein) NP_001145 NP_033803 Location (UCSC) Chr 4: 121.67 – 121.7 Mb Chr 3: 36.5 – 36.53 Mb PubMed search Wikidata View/Edit Human View/Edit Mouse Annexin A5 (or annexin V) is a cellular protein in the annexin group. In flow cytometry, annexin V is commonly used to detect ...