Tissue microarrays (TMA) construction and immunohistochemistry
This protocol is extracted from research article:
The expression and clinical significance of TPM4 in hepatocellular carcinoma
Int J Med Sci, Jan 1, 2021; DOI: 10.7150/ijms.49906

The construction of TMA covered 110 pathological sections of HCC and 10 normal samples according to the standard method 12. Continuous 5 μm portion of pathological section was removed in TMA block mass for immunohistochemical analysis, followed by staining basing on the standard procedures. Subsequently, the slices were dewaxed in xylene and rehydrated in fractional ethanol. Activity of endogenous catalase would be inhibited by 0.3% H2O2. After antigen recovery, the citrate buffer was heated with sodium for 20 mins in a self-cleavage tank for 20 mins. Sections were then incubated and 1×phosphate-buffered saline (PBS) which contained 5% normal goat serum for blocking was used for 30 mins. Sections were cultured at 4°C with primary antibody from TPM4 (rabbit polyclonal, Proteintech, China) for a night, following being washed for 3 times by 1×PBS and cultivated with HRP conjugated-secondary antibodies at room temperature. 1×PBS would be applied for additional wash after 30 mins. Under the use of hematoxylin and dehydrated before mounting, these sections were stained. Immunostaining was performed by DAB Horseradish Peroxidase Color Development Kit (Beyotime, China). Phosphate buffered saline took the place of anti-TPM4 antibody to act as a negative control.

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