[Histonet] prevent wrinkles when cutting

Mon Apr 20 16:59:58 CDT 2015

Rachel

We also run multiple antibodies under multiple parameters and there is no need or reason to cut in one ribbon that many sections.  You will still be able collect somewhat serial or the sections that you need with multiple ribbons.  You are only able to fit on the waterbath in one ribbon (depending upon the size base mold that you used ) anywhere from 6 to 15 sections.  You can place ribbons side by side on the waterbath but for me personally I find it’s a bit more difficult to pick the sections up once you have placed multiple long ribbons on the waterbath.  I prefer working with one ribbon at a time, two tops when collecting multiple sections, but that’s just me.   We work with animal tissue and tissue that may not have been processed in our lab so we do soak all of the blocks.

The other thing you need to consider is that when we section a paraffin block the section thickness is not exactly consistent, we are essentially cutting a wedge as the block warms the sections becomes thicker, so if we can control the number of sections we collect in one ribbon, place the block back on ice before we collect the next ribbon, we can then maintain section thickness better.   I would think that as you section 30 +sections in one ribbon the block is warming up and the sections are becoming thicker.  Section thickness is extremely important when comparing IHC staining results, staining intensity will change in a 1 micron difference in section thickness.

Just my two cents - and I do have images and staining intensity calculations with respects to section thickness.  This is something that we have looked at recently.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Rachel M Gonzalez
Sent: Monday, April 20, 2015 3:05 PM
To: Joelle Weaver
Cc: histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] prevent wrinkles when cutting

Hi

The long ribbons are for R&D and QA purposes. Often we test under multiple conditions 1-4 with multiple antibodies to the same tissue or multiple tissue to optimize reagents.  It quickly adds up.

Rachel

On Mon, Apr 20, 2015 at 3:28 PM, Joelle Weaver <joelleweaver <@t> hotmail.com>
wrote:

> Why are you cutting such a long ribbon? You usually only a need a 
> series of 3-4 sections even for ribbon cutting. Might be easier to 
> control if you don't try to move such a long ribbon to the waterbath. 
> Drag the shorter ribbon towards you on the waterbath. Make sure the water is not too cool.
> Face the block to full face but superficial, chill on ice for some 
> moisture, take sections while the block is still very cold. Use a 
> slow, steady, smooth stroke if doing manual cutting. Make sure your 
> embedding works well for the way you orient the block in the holder. 
> Angles work well for many tissues that are prone to wrinkling. Its 
> mostly just practice though. The more you cut, the easier it becomes 
> and usually the better you get at it.
>
>
> Joelle Weaver MAOM, HTL (ASCP) QIHC
>
>
>
>
>
> > Date: Mon, 20 Apr 2015 19:06:17 +0200
> > From: j.benavides <@t> eae.csic.es
> > To: histonet <@t> lists.utsouthwestern.edu
> > Subject: Re: FW: [Histonet] prevent wrinkles when cutting
> >
> > Hi there,
> >
> > I´m curious about the soaking thing. We have never done it in our lab.
> > Which is the purpose to do it?
> >
> > Than, after facing the blocks, we chill them in a cold plate so, if 
> > wanting to do the soaking , when should we? I guess before placing 
> > them on the cold plate, but that may cause a bit of ice formation?
> >
> > Thanks a lot for your help
> >
> > Julio
> >
> >
> > On 20/04/2015 18:40, Grantham, Andrea L - (algranth) wrote:
> > > Rachel,
> > > First off, are you chilling and soaking the blocks after you face them?
> > > Do that and see if there is a difference.
> > > Don't try to get many sections to your ribbons. Shoot for a 
> > > smaller
> ribbon (5-6) sections that are good. Cut slowly but consistently.
> > > What microtome are you using? Are you using disposable blades and 
> > > are
> they sharp? Don't expect them to cut well if you use the same blade to 
> face the blocks. If you aren't using disposables, get some! They will 
> make your life easier.
> > > You might try to find a histotech at a local hospital lab who 
> > > might be
> able to give you a hands-on lesson.
> > > Don't despair! We all sat down at our microtomes those first times 
> > > and
> suffered trying to get perfect sections. It takes practice. You might 
> make some blank blocks or blocks with tissue you can spare to practice 
> your cutting techniques. I used to do this with my students and it 
> really helped them.
> > > Good luck!
> > >
> > > Andi G.
> > > ________________________________________
> > > From: histonet-bounces <@t> lists.utsouthwestern.edu [
> histonet-bounces <@t> lists.utsouthwestern.edu] on behalf of Rachel M 
> Gonzalez [rachel <@t> gbi-inc.com]
> > > Sent: Monday, April 20, 2015 9:07 AM
> > > To: histonet <@t> lists.utsouthwestern.edu
> > > Subject: [Histonet] prevent wrinkles when cutting
> > >
> > > Hi
> > >
> > > Thursday was the first time I ever used a microtome.... I move to 
> > > a lab that does not have someone dedicated to cutting. I already miss her.
> > >
> > > I have no problems getting ribbons of 10-30 sections long but the
> pieces
> > > are half the size of the original block. I am guessing they are
> wrinkling.
> > > What am I doing wrong?
> > >
> > > Thanks
> > > Rachel
> > > Senior Scientist....
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