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immunocytochemistry


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9 replies to this topic

#1 ari_doodle

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Posted 24 April 2009 - 12:31 PM

I am growing cells on slides and then fixing them so i can stain for various proteins. Is it possible to store the fixed slides for a few days or longer until I have time to stain them or do I need to proceed to staining immediately after fixation? Thanks.

#2 yobou

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Posted 27 April 2009 - 04:57 AM

it depends upon your antigen abundance (e.g. alpha tubulin VS MAD2 at kinetochores). although some says that you can keep fixed cells at 4 oC for less than a week , you should proceed immediatly with staining procedures even though your antigen has high abundance. That what I usually do in my work

#3 scolix

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Posted 02 May 2009 - 06:06 AM

you could fix the cells and keep them in 4C for a few days. but the staining is best if done immediately. Depending on the antigen, you may or maynot get good staining.

#4 Carlton H

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Posted 08 May 2009 - 06:45 AM

I used to do this all the time... Take your fixed cells / tissue and slowly ramp them into methanol (10% --> 20% --> 40% --> 60% --> 80% --> 100% x2). Now, since you won't get freezing damage, you can store them at -20*C. Between the dehydration and the cold, there will be almost no protein activity and you can store your samples for weeks, if not months.

Note that MtOH may change the structure of some proteins and some antibodies that worked before may not work on proteins that have been stored in MtOH, however I always found that this is by far the exception, not the rule. 95%+ of the time you're okay storing in methanol.

Cheers,
-Carlton
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#5 culturelady

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Posted 14 May 2009 - 06:07 AM

From my own experience I've stained upto 2 weeks after fixing cells (in methanol) and they were still fine - they might last longer than this as well! Just check them under normal light microscope to make sure the cells look ok!

#6 umam

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Posted 26 June 2009 - 11:39 AM

I need to fix and dissect mosquito larvae, can I collect them and save them at -20 or -80 freezer till i decide to dissect them? Will saving them at 4C work better?
Thanks in advance,
-Uma

#7 yobou

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Posted 27 June 2009 - 08:47 PM

I used to do this all the time... Take your fixed cells / tissue and slowly ramp them into methanol (10% --> 20% --> 40% --> 60% --> 80% --> 100% x2). Now, since you won't get freezing damage, you can store them at -20*C. Between the dehydration and the cold, there will be almost no protein activity and you can store your samples for weeks, if not months.

Note that MtOH may change the structure of some proteins and some antibodies that worked before may not work on proteins that have been stored in MtOH, however I always found that this is by far the exception, not the rule. 95%+ of the time you're okay storing in methanol.

Cheers,
-Carlton

what about rehydration procedures? I always suffered from cell lysis after rehydration especially after ethanol fixation. NowI use formaldehyde fixation

#8 umam

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Posted 30 June 2009 - 11:22 AM

Hi,

Does anyone have any immuno-microscopy protocols that I could use to check bacterial presence in mosquito larvae?
(Suggestions for primary and sec antibody, suggestions for fixation and permeabilization etc)

Thanks,
-Uma

#9 fluffybunny

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Posted 08 October 2009 - 02:51 AM

I am growing cells on slides and then fixing them so i can stain for various proteins. Is it possible to store the fixed slides for a few days or longer until I have time to stain them or do I need to proceed to staining immediately after fixation? Thanks.


Hie ari doodle,

I grow cells on slides too, fixation using acetone at -20C. I follow the protocols from a Dako IHC handbook; they state if you keep your slides at -20C, you can keep them for 7 days; however i've tried keeping them for a month with viable results.

Quote unquote the handbook for your future reference

"Stain slides as soon as possible after preparation. If it is necessary to delay staining:
  • Store unfixed slides at room temperature in a sealed container for no more than 24 hours.
  • If a longer storage period is necessary, slides may be stored for up to seven days at 20 C or up to 30 days at 70 C.
  • Individually wrap slides with two layers of aluminum foil, securely sealing all seams. Special care is required to avoid scratching or otherwise damaging the area of cellular deposition.
  • Place wrapped slides in a plastic bag, expel excess air, and seal bag.
  • Store at 20 C to 70 C.
  • When slides are removed for staining, first equilibrate slides to room temperature for 30 minutes prior to removal from the plastic bag. In order to prevent condensation on the unfixed cells, it is important that the slides reach room temperature before unwrapping the aluminum foil.
  • Unwrap slides and proceed immediately to fixation and staining."

Reference
Farmilo, A. J. (2006). Fixation and Processing. In M. Key (Ed.), Immunhistochemical Staining Methods (4th ed) (pp. 27 31). California: Carpinteria.

#10 umam

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Posted 10 October 2009 - 06:48 AM

Thanks,
this helps!!
-uma




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