[Histonet] (no subject)

[Jack Ratliff]

Ooi,

 

As you know, working with MMA is tricky enough to have to worry about losing sections during staining. Unfortunately, I cannot speak with experience in using Bond-rite slides, but maybe there is someone monitoring this message thread that can provide helpful information on that topic. However, I can speak to the experience of using Haupts coated slides.

 

First, it is not uncommon to observe hematoxylin background staining from Haupts coated slides. Second, you are correct in that it takes just the right concentration of Haupts solution in order to retain section adherence to the slide. Given these two observations, allow me to provide a few suggestions for working with Haupts coated slides.

 

Before I prepare my slides, it is necessary to prepare a working dilution of Haupts solution. Whether or not you use in house prepared or commercially prepared concentrate, you should typically look to a ratio of 1:1 with liquified concentrate to 50% EtOH. This is a pretty standard dilution that works for me. You definitely do not want to go higher than a 50% concentration of Haupts, but you could try tweaking it a little within the 40-50% range. I would suggest that maybe you do a short experiment using different dilutions of the Haupts concentrate so that you may zero in on that one dilution that will minimize background staining and optimize section adherence. You may not have perfection with the hematoxylin background or aniline blue in a trichrome stain (I usually substitue aniline blue with SF light green yellowish), but you may find something that you can live with for the H&E.

 

Next, you might then try to work with the hematoxylin staining a bit. Specifically, maybe you can tweak the hematoxylin staining time by keeping it standard (I use hematoxylin 2 from Richard Allen and typically stain for 3 minutes) and/or increasing it a bit so that you can extend the acid clarifier step (I clarify or decolorize for 20 seconds) a little longer to help wash out or decolorize the background staining. Obviously you do not want to compromise your nuclear detail, but this should help a bit.

 

Please feel free to ask any additional questions as needed.

 

Jack Ratliff

 

 

 

 

 
> Date: Wed, 18 Mar 2009 05:33:01 -0700
> From: ooitinghuay <@t> yahoo.com
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] (no subject)
> 
> Hi, I just began to work with the methy methacrylate embedding plastic section. I am facing the difficulities to attach the sample on the glass slide.
>  
> I have tried using Haupts adhesive coated slides and also the bond-rite slide to attach my 5micron sample. I am doing normal hematoxylin and eosin staining. The section always detach from the glass slide towards the end of the staining. 
>  
> I need to use the concentrated Haupts adhesive coated slide to reduce the chance for the section from lift up from the glass slide. However, I observed the high and dirty background after the staining.
>  
> I noticed that some users are using Bond-rite slides to attach their samples. I tried this. However, the sections always lifted up towards the end of the staining. I am not too sure whether is my technique that giving me this kind of problem. I prewarmed the Bond-rite slides and put my sections in the 55C waterbath. Then I fished the section using the Bond-rite slide and lay it down on 55C heater. And finally in the oven incubator for more than 18 hours at 55C before staining...
>  
> I would appreciate if there is any suggestion that can help to attach the section on the glass slide. 
>  
> Thank you very much!
>  
> Yours sincerely,
> Ooi
> Singapore
> 
> 
> 
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