Hello Ling Zhu

If I were you, I would check the pH of the staining solution. If the pH of the staining solution drifts above 6, the reaction may be inhibited. Also, the optimal temperature for the β-galactosidase staining reaction is 37°C in a CO2-free environment since CO2 can alter the pH of the staining solution and inhibit the reaction. 

Are you sure about the incubation time? Usually, cultured cells require shorter incubation times, such as 1-4 hours, but that will again depend on the cell type and the kit used. I have mentioned this because excessive incubation time can lead to background staining or crystal formation due to pH changes. You may observe the staining under a microscope, and the incubation time may be extended, if necessary. 

So, please check on the following points.

a. Ensure the staining solution is within the optimal range (around pH 6). 

b. Make sure the incubation is performed at 37°C in a CO2-free environment. 

c. Adjust the incubation time, may be shortening it will help, if required!

Hope these suggestions help!

Best,

Malcolm Nobre Wow! Thank you for your answer. I will check the factors you mentioned above.

Lily Watson & Malcolm Nobre ,

Crystallization after dyeing may be caused by evaporation of the solution during the dyeing process. Pay attention to protection.

It is not recommended to incubate in an oven. It is recommended to use a 37℃ water bath.

If crystals are found before observation, they can be washed several times with 70% ethanol before observation. Crystallization generally has little effect on dyeing.

The answer to this question comes from MedChemExpress (MCE) Technical Support.

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