| Summary: | Microsatellite instability (MSI) accounts for the development of about 15% of all
colorectal cancers. It is characterized by mutations in mismatch repair (MMR) genes
that render cells unable to detect errors during DNA replication. Mutations in the
MMR genes, MLHl and MSH2 are responsible for the majority of CRC with MSI.
These MMR proteins can be detected by immunohistochemistry (lllC). IHC has
proved sensitive and specific when compared to molecular analysis of MSI.
Nevertheless, generally, there is no published literature as yet on studies of MSI in
Malaysia, and specifically using lliC. Before any lliC staining procedure can be
done, it has to be optimized. In this study, attempts were made to optimize rnc
staining of MLHl and MSH2 proteins using polyclonal rabbit antibody by Biovision
on formalin-fixed paraffm-embedded sections of normal colon, taken from resected
margins of colectomy specimens. Either Vectastain Elite ABC Kit or DAKO
En Vision Kit was used as secondary detection system. The staining procedure was
done using different dilutions of the primary antibodies and incubated for different
durations at different temperatures. Antigen retrieval procedures were introduced
using citrate buffer pH 6 or Tris-EDTA buffer pH 9. The results showed that
incubation of sections at room temperature using different dilutions of antibody failed
to produce any nuclear staining. Staining with DAKO En Vision was still
unsatisfactory, although background staining was reduced. Introduction of antigen
retrieval procedure using microwave did not improve results. When incubated at 4°C,
non-specific background staining was produced.
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