Author Topic: No staining of Beta-2-microglobulin or Transferrin on acetone fixed Hela cells  (Read 4595 times)

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Offline iLOVEihc

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Hello all,

I am having trouble getting any positive staining on my acetone fixed Hela cells.

Please see my protocol here:

Grow Hela cells in slide chambers
Remove media, wash with PBS
Add Room Temperature acetone and fix for 10 min
Allow to dry for 10 min
Wash with TBS
Add 3% H202 in water for 15 min at RT
Wash with TBS
Add 10% goat serum for 30 min at RT
Add primary - Rabbit anti-human Beta-2-Microglobulin, or Rabbit anti-human Transferrin incubate for 1 hr at RT
Wash with TBS
Add secondary - Goat anti-Rabbit-HRP ( this is a standard antibody from DAKO Product#http://www.dako.com/uk/download.pdf?objectid=104707002  P0448
incubate for 40 min
Wash with TBS
Add DAB substrate for 10 min at RT
Wash in TBS
Dehydrate and mount in DPX.


My antibodies were diluted in TBS only. I was wondering if my lack of staining could be due to using a single goat anti-rabbit HRP antibody rather than the Dako Envision Polymer secondary, what do you think?

Any help much appreciated!

Thanks  :)


IHClovER


Offline gula

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what does the antibody datasheet say about pretreatment of the cells or tissue?
which titer do you use with the Primary?
Do you use positive controls?

The polymer detection is more sensitiv, but you should also get some staining with simple 2-step IHC, if the antibody-titer is correct and the incubation time is
long enough.
gula

Offline iLOVEihc

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Hi Gula,
Thankyou very much for your reply.

I chose the antibodies from sigma they are Rabbit anti-Transferrin from Sigma #HPA001527
and Rabbit anti-B2M from Sigma #HPA006361.

I chose these because they had been used by the Human Protein Atlas to stain cell lines and tissues.
I chose Hela cells as according to the HPA these are positive for both Transferrin and B2M. See here: http://www.proteinatlas.org/ENSG00000166710/cell/HeLa and here:http://www.proteinatlas.org/ENSG00000091513/cell/HeLa

It says on the datasheets that they used antigen retrieval at ph6 and an antibody dilution of 1:725 for B2M and 1:500-1:1000 for Transferrrin. The secondary antibody should be used at 1:100 to 1:200.

I titrated my primaries in TBS as follows:
anti-transferrin - 1:125 - 1.6ug/ml, 1:250 - 0.8ug/ml, 1:1000 - 0.2 ug/ml
anti-b2m - 1:80 - 1.3ug/ml, 1:175 - 0.6ug/ml, 1:725 - 0.15 ug/ml 

I didn't do antigen retrieval as i didn't think this would be required for cells that were fixed in acetone and then frozen before staining... any ideas greatly appreciated.  :)




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Offline gula

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I'm from a histolab. So my way would be making a cellblock from the harvested cells in agar, fix it in formalin over night and process the cellblock to paraffin.
In this way, you can treat the cells like paraffinslides. They resist preatreatment like HIER and so on. And you can use paraffinslides as positivcontrol.

It is possible, that antibodies, that work on FFPET, don't do this on aceton fixed cells. Also Aceton influences the epitope and the antibody may fit just to the fixed epitope.

On the other side, I would increase antibody-concentration or incubate over night. Just to see, if staining occurs.
Perhaps you need a step to render the cells more permeable.

I'm no expert in working with cells. Don't know, how well they stay onto the slide during treatment....

gula

Offline iLOVEihc

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Hey Gula.

Thanks for your advice. I don't have any access to perform wax embedding.

After your comment though I have decided i will try some other fixatives, such as methanol:acetone, 95% ethanol, 10% NBF.

I think methanol:aceton combination should permeabilise the membranes, for the cytoplasmic targets.

I will also try a 0.3% Triton-X in TBS for 10 mins at RT to see if that helps!

Thanks again!  :)
IHClovER

Offline iLOVEihc

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Hello,

I thought I would give an update. I tried using acetone at -20C, 3.7% formalin or methanol:acetone 1:1, at R.T. for 10 min, and I tried permeabilisation with both 0.5% tween20 for 10 min or 0.3% triton-X doe 10 min. And still no staining! a few very faint cells stained after permeabilisation but not at all what i would expect, given the high expression that should be present....  :(

I am using TBS only as an antibody diluent and am now considering putting 0.05% tween20 in there, plus adding a permeabilisation with tween20, but i am starting to think these antibodies will just not detect the epitope in my tissues!! for whatever reason....

 
IHClovER