Be carefull when tranfering your slides to low level.

Some protocols are describing fixation of (BCG or avirulent strains with sodium azide or glutaraldéhyde. But for virulent strains of M. tb this is not safe.

We used Paraformaldéhyde at 4 % for 1 H incubation:

- Wash the coverslips with PBS

-Incubate with PBS containing 4% w/v PFA for 1 Hour. After fixation, all

subsequent steps can be performed on the bench.

-Wash the cells twice with PBS.

-Incubate with NH4Cl 50 mM in PBS for 15 minutes.

-Wash the cells once with PBS.

-To permeabilize the fixed cells, 0.1% w/v triton in PBS for 5 minutes.

-Wash the cells twice with PBS.

-Incubate the cells with 1% w/v bovine serum albumin (BSA) in PBS for 15 minutes.

-Incubate the cells with the desired antibodies

Youcan also use PFA at 2 % if youare analysing a sensitive organelles or compartments. Formalin also is used.

Good luck

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC87817/