Try this:

mix 14.6ml acetic acid with 485.4ml dH2O and solve 1g benzidine dihydrochloride (keep this in dark, e.g. covering in foil)

For staining: mix 1ml of this mixture with with the same volume of cell suspension and add 1µl H2O2 (2%). Incubate for 1-2min at rt and after incubation you can measure your probes.

Try this:

mix 14.6ml acetic acid with 485.4ml dH2O and solve 1g benzidine dihydrochloride (keep this in dark, e.g. covering in foil)

For staining: mix 1ml of this mixture with with the same volume of cell suspension and add 1µl H2O2 (2%). Incubate for 1-2min at rt and after incubation you can measure your probes.

This is how I got it to work:

Benzidine working solution: 

Prepare stock 0.2% tetramethylbenzidine (TMB) (Sigma, # T8768) in 0.5% acetic acid:

1. Dilute acetic acid 1:200 (1mL acetic acid in 199mL H2O)

2. To prepare a 0.2% TMB, dissolve 2mg/mL TMB in 0.5% acetic acid

Pr 50mL: Dissolve 100mg in 50mL 0.5% acetic acid. This can be stored @ 4C for up to 1 month.

3. Prepare just before use: Add 5ul of 30% hydrogen peroxide/1mL of 0.2% TMB.

Pr 10mL: Add 50ul of 30% hydrogen peroxide

Staining:

Wash K562 cells once with PBS and resuspend in 0.25-0.5 mL of PBS. Add 1 volume (0.25-0.5 ml) of 0.2% tetramethylbenzidine working solution and incubate 10-30 minutes in the dark at room temperature (it’s toxic, so do not incubate longer than 30 min in total including picture taking). Count brown cells in light microscope

Thank you both.

 Cathrine,

How did you get TMB to fully dissolve in 0.5% acetic acid? I ordered the same TMB and it won't dissolve. I even tried carefully heating it up briefly.

For actual reason only:  pointing to another Q&A regarding Benzidine stain for hemoglobin/haemoglobin and measuring using an hemocytometer/haemocytometer...: 

by Sahil Mehta,  Washington University in St. Louis

"Does anyone have a protocol for Benzidine Staining of Frozen Tissue Sections?"

I will be using this staining method to measure Heme in mouse lung, liver, and spleen tissues,   to be found at:

https://www.researchgate.net/post/Does_anyone_have_a_protocol_for_Benzidine_Staining_of_Frozen_Tissue_Sections?

Regards, W.M.