[Histonet] Re: iliac artery attachment to slides?
ratliffjack <@t> hotmail.com
Tue Aug 30 15:57:50 CDT 2011
I agree with Teri regarding the Haupt's Adhesive and assume that when you mean a gelatin coating that it is possible that you might already be using this product based upon past conversations. If you are using Haupt's, then I am confused that you are having difficulty with your sections remaining attached to the slide. I am in that group Teri mentioned that swear by Haupt's and it ability to retain sections throughout staining. This then leaves me to question the use of 2-MEA for removal of the resin since you seem to be having problems after this step.
Please remember a few things (my opinion and I welcome the challenge from others) when mounting resin sections to gelatin coated slides:
#1 - The concentration of gelatin in the coating solution is directly proportional to the adhesive property of the section to the slide.
#2 - The concentration of the gelatin in the coating solution is directly proportional to the background staining of certain high molecular weight dyes (i.e. hematoxylin, Aniline blue, etc.).
#3 - The adhesive property of the gelatin, attachment of the section to the slide, is proportional to the heat activation of the gelatin during the slide section drying phase (i.e. heating in an oven for a period of time at a certain temperature range).
Allow me to briefly expand upon this last statement. The best way that I can explain all of this to people is what I simply call the "tape effect". If you use regular Scotch tape on paper in a room temperature environment, 8 or 9 times out of ten you can safely remove the tape without tearing the paper and without a sticky residue. If you repeat the same procedure in a warmer environment, like say you left it on the paper and stuck it to a window inside your car with all the windows up on a hot summer day, you will be able to remove the tape while it is hot without damage to the paper, but you will leave a very sticky residue on both the paper and the window due to the heat activation of the adhesive. However, if you wait until late evening to perform the same task after the tape has cooled, the adhesion is much stronger and you are highly likely to tear the paper and find it difficult to remove easily from the window.
This is how I explain heat activation of the gelatin adhesive and it is for this reason that I use an aluminum slide press as a "heat sink" to help dry ALL slide sections evenly and activate the adhesive properties of the gelatin by increasing the internal heat within the slide chambers. Essentially, you are looking for the correct proportion of adhesive concentration to background staining, all while avoiding the melting of the plastic section covers but still so that all slides dry evenly and so that enough heat is achieved to activate and optimize the adhesive properties of the gelatin (Haupt's Adhesive) coated slides. I then deplastify in warmed xylenes at 50 C, three fresh changes for 30-60 minutes depending upon the overall size of the sections (i.e. small sections on 1x3 slides or large sections on 2x3 slides) and do not lose sections!
One last thing to consider is that the geometry of your tissue (circular) is also a contributing factor because of the shrinking and swelling effect in using organic solvents like xylenes to deplastify, ethanol's of decreasing concentration to hydrate the sections, water based solutions for staining and then dehydrating again to coverslip. The density of the tissue then also increases this shrinking and swelling effect. For example, this is not as noticeable with undemineralized bone.
So, what do you do? Well, obviously you have to identify where your weaknesses are and then start changing the variables one-by-one until you optimize the method. If it was me I would eliminate the 2-MEA first, then ensure adequate and even drying of the slides as also related to adhesive activation in the oven. If you are still having problems, play with the concentration of the gelatin adhesive and learn to live with the background. There really is a lot of directions to go here, but by no means throw in the towel and give up! Hope I have been helpful and not too confusing. :)
Simple concept I know, but think about this, the proper balance of gelatin concentration, heating and cooling during the drying phase, subsequent use of chemicals, and the density properties of the tissue, all contribute to the overall section adhesion throughout staining.
> From: TJJ <@t> stowers.org
> To: histonet <@t> lists.utsouthwestern.edu
> Date: Mon, 29 Aug 2011 13:18:32 -0500
> Subject: [Histonet] Re: iliac artery attachment to slides?
> Hi Jim, sounds like you are having a time of it.
> I figure Jack Ratliff will chime in as soon as he sees this. In the meantime I will give you the same advice he gave to me.
> If you are having troubles with tissues adhering, try Haupt's adhesive. You can find recipes on the internet to make it yourself, or you can buy it commercially ready to use (www.dornandhart.com). I have heard from several people who use this consistently with their MMA and they swear by it. Have you tried stretching the sections using a few drops of 50% alcohol and a couple of soft brushes prior to covering in plastic and clamping? It's going to be tough getting circular tissues wrinkle free. I hope others with more experience than me will chime in on this.
> Best wishes,
> Teri Johnson, HT(ASCP)QIHC
> Head, Histology and Electron Microscopy
> Stowers Institute for Medical Research
> Kansas City, MO
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
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