It sounds like you are trying to culture primary BAL cells. If you are having trouble getting the cells to stay on the slide surface, there are several things to consider. First, are you culturing on glass or plastic? If the cells aren't sticking to one, they may need the other surface. Secondly, some cells need more than just poly-L-lysine or poly-ornithine to bind to the surface. Try preparing the slides by incubating the slides with first lysine or ornithine for an hour at room temp, remove and allow surface to dry. Next, incubate the slides with an extracellular matrix protein such as laminin or collagen overnight at 4 degrees C. Remove the laminin/collagen immediately prior to plating the cells making sure that the surface does not dry. The next thing to consider is what type of media and supplements to use in order to promote growth. Dialing that in correctly will go along way to maintaining cells health and thereby increasing the overall number of cells. I usually fix my primary cells for a very short period of time (20-30 minutes) in 4% paraformaldehyde. I rinse gently in PBS and proceed to labeling. Unlike alcohol based fixatives, paraformaldehyde causes very little tissue shrinkage.

It sounds like you are trying to culture primary BAL cells. If you are having trouble getting the cells to stay on the slide surface, there are several things to consider. First, are you culturing on glass or plastic? If the cells aren't sticking to one, they may need the other surface. Secondly, some cells need more than just poly-L-lysine or poly-ornithine to bind to the surface. Try preparing the slides by incubating the slides with first lysine or ornithine for an hour at room temp, remove and allow surface to dry. Next, incubate the slides with an extracellular matrix protein such as laminin or collagen overnight at 4 degrees C. Remove the laminin/collagen immediately prior to plating the cells making sure that the surface does not dry. The next thing to consider is what type of media and supplements to use in order to promote growth. Dialing that in correctly will go along way to maintaining cells health and thereby increasing the overall number of cells. I usually fix my primary cells for a very short period of time (20-30 minutes) in 4% paraformaldehyde. I rinse gently in PBS and proceed to labeling. Unlike alcohol based fixatives, paraformaldehyde causes very little tissue shrinkage.

It depends on the antigen you are trying to preserve.  Paraformaldehyde reduces detection of some antigens.  For example, fixation of cryosections of T-cells in paraformaldehyde for more than a 30 seconds eliminates/reduces detection of CD4 antigen. Also, paraformaldehyde shrinks cells whereas acetone does not.  If paraformaldehyde is needed to preserve the antigen, fixation by acetone followed by 2% paraformaldehyde for less than a minute gives good morphology and usually preserves antigenicity. One needs to experiment first. Also one can place the cells on superfrost + slides, dry for 30-60 min, then fix appropriately as described above. 

First we have to know what cells are we talking about; BAL might be made of  different type of cells depending of the state of the mice (no treatment or treatment, KO that lead to cell infiltration and inflammation....ect) : The BAL from  untreated mice is made of 99% alveolar macrophages, however after infection the BAL is made predominantly  of neutrophils. If you are using BAL from untreated mice  then  then you could plated them for about 1h:30 min in slide chamber, fix with 4% paraformaldehyde for 10 min and you are good to go. The other thing you could do also is cytospin, in this case will have a heterogeneous population, and again fix with acetone (as mentioned above or PFA  (I usually do 10 to 15 min no more.

After collection of the BALF you can spin the cells on superfrost plus slides (pre cleaned slides of thermo) (or you can use poly-L-lysine coated or APES-coated slides...), dried for 15-30 min (RT) and then can be fixed in cold Aceton (-10 to -20) for 10 min., or in 4% PFA, dried for 30min.

I think, the cell adhesion depends on the the coating method of the slides, not on fixative method.

The best protocol would be a cytospin, see protocol here(http://www.ihcworld.com/_protocols/histology/cytospin.htm), using adherent slides (cover with silane). It will not dramatically affect the morphology of the cells.

For the fixation I would use 10 min incubation in cold acetone, and left dry the slides. with this fixation you will not affect so much the morphology neither the antigens in your cells.

best

You can fix BAL cells in paraformaldehyde that reduces detection of antigens. or fix cryosections of T-cells in paraformaldehyde 1 minute atleast. It eliminates/reduces detection of CD4 antigen. You can use  acetone to avoid shrinkage. Paraformaldehyde is needed to preserve the antigen, fixation by acetone followed by 2% Paraffin-embedded sections of whole lungs can be stained with MCP-1 polyclonal antibody (ab7202) or rabbit polyclonal IgG (ab27478), by using the Envision detection system (Dako, Cambridge, UK),  Antigen retrieval can be  performed using “target retrieval solution” (citrate buffer; Dako) for 20 min in the microwave. BAL cells from FACS-sorted populations can be stained with CD11b, Ly6C, and Ly6G were cytospun onto glass slides and stained with hematoxylin solution for 2 min.

For immunohistochemistry, I would make a cell suspension in 1 - 10% of a non-cross reactive serum (for most application FCS will do)

I would fix cells either in cold aceton or by rapid drying in the air at 20 - 37 degrees, storing the slide dry. 

I would not use paraformaldehyde as it might reduce immunodetection by many antigens. If your antibody is unaffected, of course, this would not be a problem.

Dear all, 

Thank you for your suggestion. If possible, I would like to ask for more details. After coating and fixing, how can we preserve cell slides prior to immunostaining? And for how long? 

I'm trying with air drying method after cytospin BAL cells, following by quick fixation in 4% PFA for 10min, and then air drying. Hope that while protecting the antigen, our cells would stick firmly to microscope slide (we use super frost plus slide). 

Dear anh Dung, Rafael, Hall and Jacobs, 

We are working with cysteinyl leukotriene and purinergic receptors. Do you know whether PFA may affect those antigens? How can we know whether our antigens would be affected or not (based on their characteristics, for ex)?

Regarding to other relevant studies, some use either paraformaldehyde or acetone for fixing cells and tissue. So, I just tried only with paraformaldehyde up to now, since we didn't have acetone. Thank you.  

Dear Ravi Kant Upadhyay, 

Thank you for your suggestion. We also use paraffin-embedded section. Usually, we fix tissue in 10% formaldehyde. Do you know whether it caused tissue shrinkage or reduction of immunodetection? Thank you

I do not know bout paraformaldehyde on specific receptors, the effects will be dose, time and antigen related.

In the absence of acetone I have used air drying at room temperature mildly heating (e.g. 35 C) for fixation, without observable loss of antibody reactivity. Slides are also best stored in  dry environment.

Thank you Jacobs, I will try your protocol

in a situation where someone does not have cytospin to spin the cells, what other alternatives can I use.

@Ibrahim: How about using Polylysine-L coating method? To my knowledge, you can use either Polylysine-L-coated slides or Polylysine-L-coated coverslips. It's simple, and you may find many available protocold. Hope this help!