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Author Topic: ICC cytospins of nasal epithelial cells  (Read 6447 times)

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Offline Eugene

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ICC cytospins of nasal epithelial cells
« on: March 11, 2009, 07:01:19 PM »
Hello
Sorry Im a newbie to ICC etc.  My protocol involves cytospinning asthma patient nasal epis onto slides and I want to fluro Ab label them (easier to see vs. HRP etc?) for an intracellular protein (XIAP aka BIRC4).  I would like to know if the protocol Im going to use is valid and I have a few quns along the way.  Thanks for anyone that can help :)

Procedure...
1. obtaining nasal airway epis form patients (brushings up the nose that you vortex off in PBS)
2. cytospinning 100 uL of 50 000 onto a slide at 1500 rpm for 20 mins --> air dry for 30min (when I do this with cell lines I get fibres on the slie - should I wash the funnel first or something?)
3. I was going to acetone fix from here simply becasue this will also permeabilise the cells for XIAP localisation : -20*C acetone immersed for 5-10 mins (how long is too long? what of methanol or 1:1 both? what of others such as g.acetic acid, methanol, ethanol... is there a selection criteria?)
4. rinse 3x in PBS - can they be batched at this stage in the fridge etc?)
5. block with 2-5% sera (is this FCS or what the 2* Ab is suspended in?)
6. incubate with R&D Systems pAb to XIAP at 1:50 for 1 h at RT with gentle rocking in PBS (is PBS ok? incubate at a lower temp?)
7. incubate with FITC-labelled (I heard green is good) 2* Ab for 1 h at RT in PBS
8. wash 3x in PBS
9 sghould I fix the cells again now in 3.7 % Formaldehyde? I want to counter stain with DAPI of K167 for nuclear localisation too - will formaldehyde prevent this?  If so can I keep these in the dark at 4*C and come back to them later and counter stain then?

What am I forgetting?

Thanks to anyone who can respond.
Eugene

ICC cytospins of nasal epithelial cells
« on: March 11, 2009, 07:01:19 PM »

Offline CanuckPhD

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Re: ICC cytospins of nasal epithelial cells
« Reply #1 on: March 12, 2009, 03:15:34 AM »
My sugegstions are below in your protocol

1. obtaining nasal airway epis form patients (brushings up the nose that you vortex off in PBS)
2. cytospinning 100 uL of 50 000 onto a slide at 1500 rpm for 20 mins --> air dry for 30min (when I do this with cell lines I get fibres on the slie - should I wash the funnel first or something?) I have never had an issue with fibers on the slide from the filter, so can't make a suggestion here. Just make sure that everything is cleaned including your slides
3. I was going to acetone fix from here simply becasue this will also permeabilise the cells for XIAP localisation : -20*C acetone immersed for 5-10 mins (how long is too long? what of methanol or 1:1 both? what of others such as g.acetic acid, methanol, ethanol... is there a selection criteria?) 5 minutes in acetone is usually okay, however different antigens will require different fixations. You must perform experiments/trials to determine this. I suggest if you have a cell line expressing this protein try different conditions on this before you use your human samples.
4. rinse 3x in PBS - can they be batched at this stage in the fridge etc?) If you want to batch your slides then place them in the -80 after you dry them
5. block with 2-5% sera (is this FCS or what the 2* Ab is suspended in?) It is usually serum from the same species that your secondary antibody is produced in. Using 0.05% Tween-20 is good to permabilize your cells.
6. incubate with R&D Systems pAb to XIAP at 1:50 for 1 h at RT with gentle rocking in PBS (is PBS ok? incubate at a lower temp?) for 1 hour RT is fine, I personally use TBS instead of PBS as I seem to get better staining
7. incubate with FITC-labelled (I heard green is good) 2* Ab for 1 h at RT in PBS
8. wash 3x in PBS
9 sghould I fix the cells again now in 3.7 % Formaldehyde? I want to counter stain with DAPI of K167 for nuclear localisation too - will formaldehyde prevent this?  If so can I keep these in the dark at 4*C and come back to them later and counter stain then? just DAPI stain no need to fix again

Offline Eugene

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Re: ICC cytospins of nasal epithelial cells
« Reply #2 on: March 13, 2009, 02:06:13 AM »
Hi CanukPhD

Thanks for that, I'll do as you suggest. I'll let you know how I went on my cell lines in a couple of weeks, when no doubt I will have some more specific questions.

Cheers
Eugene

Re: ICC cytospins of nasal epithelial cells
« Reply #2 on: March 13, 2009, 02:06:13 AM »