2 replies to this topic

[Maya1988]

Hi all,

Having a forum like this is really great and I am happy I could join - thanks to the team!

I have a question regarding immunohistochemistry for insulin and I am pretty desperate. I have the following protocol to stain insulin positive beta-cells for stereology, but somehow I can't get a strong signal (eg strong enough to take decent pictures of my slides and do some consistent counting). Info: The pancreas is collected, placed in 4%PFA overnight, then in 70% etOH for at least 24h and then embedded in paraffin.

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Deparaffinization:
Xylene I, II, III, IV - each 6 mins
Ethanol 100% I, II, III - each 6 mins
Ethanol 90%, 70%, 50%, 30% - each 6 mins
1 minute of running tap water

Block
PBS-T with 5% serum from species in which secondary antibody was raised - 20 min at RT

1ary AB
2% serum block in PBS-T with 1:50 primary antibody (guinea pig anti insulin)

3 min wash in PBS-T

2ndary AB
2% serum block in PBS-T with 1:40 secondary antibody (rabbit polyclonal anti guinea pig IgG, with green fluorescence)

3 min wash in PBS-T

mount and fix with Vectashield+DAPI
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Ok so first I thought the problem was the embedding of the tissue itself, thus the many steps of deparrafinization and rehydration. This improved the overall signal strength. (I mainly did this because I noticed that when I do the whole protocol TWICE for a set of slides, then they look decent enough to count.)

Well so I don't want to repeat the whole protocol for each set of slides I do, as this costs time and reagents. Does anyone see an obvious flaw in the protocol?
Does anyone have the same problems with consistency in such protocols? I literally treat every slide the same way, process in the same amount of time, eg. I don't do too many slides at once, they don't dry out...and STILL it happens that slide a looks good but slide b shows nearly no staining at all...

I am really desperate as I have been trying to optimize this protocol for a while now,
please any help is welcome!

Many thanks,
best wishes,
Maya

[bob1]

Sorry this is late, I can't see any flaw in the method, other than that you may need a permeabilisation step (10 min in 0.1% triton-X100), you could try primary incubation for longer and try diluting the secondary more - typical 2ry concentrations are in the range of 1:1000 - 1:10000.

You are doing the secondary and later steps under dim light or in a dark room to prevent photobleaching I take it?

[Maya1988]

Hi bob1,

Thanks for your help and sorry for not getting back to you earlier, yes I did those steps in low light/in the dark.
I think I have found the problem, it must have had to do with the embedding of the tissue itself. I increased the deparaffinization and rehydration times and some of the samples I had to rehydrate twice. We have had the embedding done at a central facility and I suspect it was not 100% cleanly done, since I've also had trouble with ISH and am now doing my own embedding!

Best,
Maya