Immunostaining cultured cells

We also conducted experiments to compare our culture method with the standard neurosphere culture. In the standard neurosphere culture, cell number increased approximately nine times over 5 days. Immunostaining showed that the neurosphere cultures contained 54 5.3% nestin+ cells and 41 7.4% nestin+ pIIF cells. This demonstrated that the standard neurosphere culmring method was less efficient than EGF-immobilized substrates for selectively expanding NSCs. Thus, the EGF-immobilized substrates prepared from mixed SAMs with 10% COOH-thiol provided the most efficient method for selective NSC expansion. 

Raats J. M. and Hof D., Recombinant antibody expression vectors enabling double and triple immunostaining of tissue culture cells using monoclonal antibodies, Eur. J. Cell. 

Some types of cultured cells, such as Drosophila Schneider cells, can be grown directly on glass microscope cover slips. Such cells can be subjected to FISH procedures as easily as they can be immunostained. Here is a simple procedure for fixation ... 

FIGURE 6.14. Effect of pretreatment with 1% sodium dodecyl sulfate (SDS) on immunostaining of cultured epithelial cells with anti-AE1/2 affinity-purified antibody at a final concentration of 0.45 i,g/ml. (A) In the absence of SDS treatment, AE2 is barely detectable in MDCK cell cultures. (B) After SDS treatment a bright basolateral membrane staining is visible. Reproduced, with permission, from Brown et al. (1996). Copyright 1996 Springer-Verlag. 

The cell suspension is now ready to introduce into tissue culture, immunostain, or deposit onto a microslide with a Cytospin. 

Fig. 7. Identification of apoptotic cells based on the immunocytochemical detection of the 89-kDa PARP cleavage fragment. HL-60 cells untreated (A) or treated with camptothecin (B) (refs. 26,28) were immunostained with FITC-anti-PARP p98 and PI according to the protocol. Note that in the treated culture, S phase cells preferentially were undergoing apoptosis. Some cells have diminished ( sub-G ) DNA content, likely due to shedding of apoptotic bodies and/or extensive DNA fragmentation.

Fig. 1. Cultured RBL-2H3 cells unpermeabilized (a) and permeabilized with acetone (b) for 5 min at -20°C. Without permeabilization, there is no immunostaining. Flowever, after permeabilization, the antibody has access to the antigen and the cells immunolabel.

---