Author Topic: Tissue keeps falling off slides during H&E staining, need help ASAP.  (Read 3860 times)

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Offline SRajpara

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Hello all,

I'm having trouble keeping tissue on the slides while I do H&E staining. I couldn't add this in the subject title but I also had trouble because the counterstain came out much brighter than the Hematoxylin. I did one yesterday and followed this protocol:

-Take slides out of -20C freezer and let it air dry for 30min
-Dehydrate with 100% EtOH for 6 min
-2 min incubation in Hematoxylin
-rinse with ddH2O
-2 min incubation in tap water
-8x dip in acid EtOH (4mL of 6N HCl in 200mL of 70% EtOH)
-2 min incubation in tap water
-5x dip in base (3.08g Lithium Carbonate in 200mL of ddH2O)
-2 min incubation in ddH2O
-30 sec incubation in Eosin Y
-5 min dehydration in 70% EtOH
-15 min clearing in Citrisolv

I'm working with kidney, spleen, adipose, liver and skin tissue all from adult male rats. The reason why I cannot bake these slides is because I treated the rats with polystyrene microspheres, and they will melt if I bake the slides at 55C. I'm also using SuperFrost Plus slides, and using OCT as the embedding compound.

Please advise as to what I should do in the near future. Thank you.

SR


Offline gula

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Re: Tissue keeps falling off slides during H&E staining, need help ASAP.
« Reply #1 on: March 03, 2015, 11:14:55 AM »
The first thing, that comes to my mind is the question: why do you dehydrate your slides at the beginning?
Hematoxylin is usually aequous and after a certain time of air-dry you can go directly into hematoxylin. I would skip the dehydration step.
What Eosin are you using? aequous or alcoholic? For aequous Eosin you can introduce additional rinsing steps in dd H2O (or tapwater). For alcoholic Eosin you can also prolong rinsing in diluted ethanol. What is the Eosin's concentration. We use 2%.
If you have the impression, that the hematoxylin-staining is too light, prolong the time in hematoxylin and be carefull with the differentiation-step in acid-ethanol (you can also use acidified ddH2O).
gula

Offline SRajpara

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Re: Tissue keeps falling off slides during H&E staining, need help ASAP.
« Reply #2 on: March 03, 2015, 12:37:44 PM »
I dehydrate the slides beforehand to make sure the tissue stays on the slides. We use aqueous Hematoxylin and Eosin. We also use 2% Eosin. So are you saying I should skip the first alcohol step and go straight to Hema? Thanks

Offline gula

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Re: Tissue keeps falling off slides during H&E staining, need help ASAP.
« Reply #3 on: March 03, 2015, 01:00:27 PM »
There is no scientific reason, why dehydration should improve the sticking of the sections. In my opinion it renders the section more brittle and may lead to shrinking of the section. So the chance to loose the section will be higher, when it doesn't adhere to the glass from the beginning.
good luck
gula

Offline SRajpara

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Re: Tissue keeps falling off slides during H&E staining, need help ASAP.
« Reply #4 on: March 05, 2015, 04:29:51 PM »
Ok so I did the H&E again, and tissue actually fell off between the acid ethanol step and before the base step. For the acid ethanol formulation, we use 4mL of 6N HCl and 200mL of 70% ethanol. Base is 3.08g of Lithium Carbonate in 200mL of double distilled water. i'm not sure what's going on, and I'm starting to get frustrated with this protocol. Any advice will be much appreciated. Thank you.

SR

Offline gula

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Re: Tissue keeps falling off slides during H&E staining, need help ASAP.
« Reply #5 on: March 07, 2015, 03:33:41 AM »
adhesive slides; grease-free, clean slides
acidified water instead of acid ethanol. = dest. water (ca. 100 ml) plus 2-4 drops of 100% acetic acid. The pH of the acidified water is about 5-6, not really very acid. The differentiation may take longer,but is more gentle.

The thinner the section, the better the adherence. How thick are your sections?
gula


Offline SRajpara

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Re: Tissue keeps falling off slides during H&E staining, need help ASAP.
« Reply #6 on: March 08, 2015, 03:00:47 PM »
I apologize for the delayed response, but I use Super Frost Plus slides (is that not adhesive enough?) and my sections are 20microns for this project, but 8microns for the kidney and spleen sections, which have only had tissue folding over. I will use the weak acid formulation you gave me on my next try tomorrow. Thank you!

SR

Offline gula

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Re: Tissue keeps falling off slides during H&E staining, need help ASAP.
« Reply #7 on: March 09, 2015, 06:31:37 AM »
If nothing really helps in the end, you can try to stain the thick sections free floating. The sections are brought from one coplin jar to the other by mounting them on a glass slide with the help of a painting-brush. In the next jar the section is released again into the liquid.
gula

Offline SRajpara

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Re: Tissue keeps falling off slides during H&E staining, need help ASAP.
« Reply #8 on: March 10, 2015, 09:13:17 PM »
I tried the protocol today with Scott's Tap Water Substitute (I made it myself) and with the weak acid formulation, and it worked really well. I have yet to image these, but I'm very happy with the result. Now to my IHC problems! Thank you.

Offline gula

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Re: Tissue keeps falling off slides during H&E staining, need help ASAP.
« Reply #9 on: March 11, 2015, 04:19:21 AM »
I'm happy for you  ;D.
gula

Re: Tissue keeps falling off slides during H&E staining, need help ASAP.
« Reply #9 on: March 11, 2015, 04:19:21 AM »