TMB Horseradish Peroxidase Color Development Solution for Immunohistochemistry or Blotting

Precautions：TMB is irritating. Please take effective measures to avoid inhalation or direct contact with skin.This product is a colorless to slightly blue transparent solution. Discard it when it turns turbid or darker blue.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.Instructions for Use：1. For immunohistochemical assays:a. After incubation with HRP-labeled antibody, wash 3-5 times with an appropriate wash buffer for 3-5 minutes each.b. Remove the wash buffer and add 100μl of TMB Substrate Solution dropwise.c. Incubate at room temperature in the dark for 3-30 minutes or longer (e.g., overnight in a wet box) until the desired color intensity is achieved.d. Remove the TMB Substrate Solution, add deionized water or ddH2O and incubate for 10 minutes to stop the reaction.e. Photograph directly or after proper mounting. If necessary, Neutral Red Staining Solution (, C0123) can be used for counterstaining.2. For Western blot membranes:a. After incubation with HRP-labeled antibody, wash 3-5 times with an appropriate wash buffer for 5 minutes each.b. Remove the wash buffer.c. Add an appropriate amount of TMB Substrate Solution to cover the membrane completely.d. Incubate at room temperature in the dark for 3-30 minutes or longer (up to several hours) until the desired color intensity is achieved.e. Take pictures directly.FAQ：1. Too dark backgrounda. If the background of negative control is too dark, attempt other appropriate blocking buffers or block with 10% serum from the same source as the primary antibody. Alternatively, use secondary antibodies that have been properly adsorbed to reduce non-specific bindings of secondary antibody.b. Reducing the color development time or the concentration of secondary antibody can be considered. Using other wash buffers with stronger strength or extending the washing time may also help.2. No color or too weak colora. Increase the concentration of the primary or secondary antibody appropriately. To test the effectiveness of the secondary antibody, add a drop of diluted secondary antibody to a centrifuge tube and check if the colour can be developed normally.b. Use a more sensitive detection system with higher amplification efficiency, such a biotin detection system.c. Extend the color development time appropriately.d. Use a primary antibody or ELISA kit with better effects.