Is there a significant non-specific background difference between imported and domestically produced HRP secondary antibodies in immunohistochemistry?

Domestic secondary antibodies have a high background, so don't rush to return them for now. Reducing the dilution of the secondary antibody by 1-2 times, such as using imported 1:500 and domestically tested 1:1000, 1:2000, or 1:4000, may improve the signal-to-noise ratio. The blocking solution can be replaced with 5% normal goat or horse serum and 1% BSA, and blocked overnight at room temperature or 4 degrees Celsius. Wash with PBS+0.05% Tween-20 and increase the number of washes. The inactivation of endogenous enzymes should be sufficient, but avoid structural damage caused by peroxidation. Every time a new batch number is changed, the gradient is redone. The difference between domestic batches is sometimes greater than that of imports, so titration cannot be omitted.

I have tried domestic HRP secondary antibodies from several vendors that commonly serve pathology applications, and the background varies considerably. When selecting a supplier, consider three points: whether they provide a complete COA, whether they can provide small samples from three lots, and whether they have paraffin IHC data from actual users. You can first contact domestic pathology-focused lines such as Zhongshan Golden Bridge and Servicebio to apply for trial samples, but do not rely on brand alone; you need to check the specific catalog number. For lot-to-lot stability validation: randomly divide the same section into three groups, and use each lot of secondary antibody to stain the same target, then compare background and signal intensity; if the lot-to-lot CV is large, it is not recommended for long-term projects. Only after small-scale trials show no problems should you proceed to batch replacement.

For domestic HRP used in IHC, I generally choose an RZ above 3.0, but it is not the only indicator. RZ only reflects the partial purity of HRP; nonspecific background from secondary antibodies also comes from antibody cross-reactivity, labeling rate, IgG removal purification, etc. Before purchasing, ask the manufacturer to provide: HRP RZ, SDS-PAGE purity, antibody species cross-adsorption information, and actual IHC images. If the RZ is below 3.0 and the antibody purity is average, the background risk will be high. It is recommended to first request a small pack or free trial, and compare it with the original imported secondary antibody using the same batch of sections and the same conditions.

After replacing the domestic secondary antibody, non-specific staining of the cytoplasm was performed. Two controls were first made: slices were not incubated with the primary antibody, and only the same concentration of domestic secondary antibody was added; Slice and incubate with primary antibody but without adding secondary antibody. If only adding secondary antibodies has background, it may be due to the secondary antibodies themselves or endogenous peroxidase; If there is no background without the addition of secondary antibodies, it indicates a problem with primary antibodies or endogenous enzymes/blocking. It is recommended to use 3% H2O2 methanol to inactivate endogenous enzymes in paraffin sections, and to use homologous serum with secondary antibodies for blocking solution. Wash and add 0.05% Tween-20. Domestic secondary antibody dilution usually requires re titration, and the original imported dilution may not be applicable.