[Histonet] HELP! Need some old fashioned histology advice

[Mejia, Mary]

Hello Patsy,

Thank you very much for responding!  Yes & of course, I'll be removing the celloidin from each section - we normally
do this on smaller human whole brainstem sections using ether/100% EA 1:1 - 3x - 3 minutes each with agitation.  The
latter type of sections are very easy to work with, however these future big boys I'll be getting is another thing.

Our lab is currently alcohol processing a human whole brain & after the last 95% EA - it will be embedded in 2% celloidin
& placed inside a very large desiccator under 20 psi pressure.  This part like every step will take some period of time,
I need to test several different IHC methods & hope one will actually work.

If you have any further ideas or thoughts on this subject - shoot me an email.  Thank you again for responding.

Maria
________________________________
From: Patsy Ruegg [pruegghm <@t> hotmail.com]
Sent: Sunday, January 04, 2015 7:03 PM
To: Jay Lundgren; Maria Mejia
Cc: Histonet <@t> Lists. Edu; Mejia, Mary
Subject: RE: [Histonet] HELP! Need some old fashioned histology advice

I have done something similar to this but I used tissue that was fixed but not processed and embedded, this is called enblock labeling, I infiltrated the fixed tissue with the IHC reagents, in a vial/tube, the blocking reagents, then the antibody, then the detection reagents and DAB, then dehydrated the tissue.  I used vials or tubes on a platform shaker and would infiltrate reagents for days, then after it was done I infiltrated and embedded the tissue in glycol methacrylate (GMA) so that I could section it, it actually worked.  The tissue was already IHc LABeled so all I did to the 5 micron sections after they were cut was a hematoxylin counterstain, this was mineralized bone so I had to embedd in something hard like GMA to section.

Will you remove the Celloidin before trying to do the IHC staining?  100 micron sections might be easy to float/handle using a glass pipette for transferring.  Sounds like an interesting project, good luck and feel free to ask for advise and keep us posted on your progress.

Cheers,
Patsy

Patsy Ruegg, HT(ASCP)QIHC
Ruegg IHC Consulting
40864 E Arkansas Ave
Bennett, CO 80102
H 303-644-4538
C 720-281-5406
pruegghm <@t> hotmail.com



> Date: Sun, 4 Jan 2015 11:58:38 -0600
> From: jaylundgren <@t> gmail.com
> To: mbmphoto <@t> gmail.com
> Subject: Re: [Histonet] HELP! Need some old fashioned histology advice
> CC: histonet <@t> lists.utsouthwestern.edu; mary.mejia <@t> ucsf.edu
>
> I can help with the old fashioned advice:
>
>
> - 1 scant teaspoon simple syrup
> - 2 dashes Angostura Bitters, plus more to taste
> - 1 half dollar–sized slice orange peel, including pith
> - 2 ounces good-quality rye or bourbon
> - 1 maraschino cherry
>
> As for the Histology, is there any reason you cannot mount the
> sections onto glass slides? When I was working at Genentech they were
> cutting frozen sections through whole rabbits and mounting the sections on
> (giant) glass slides. I think that rolling the tissue up, inserting it,
> and then removing it from a glass tube would destroy the tissue.
>
> Sincerely,
>
> Jay A. Lundgren, M.S., HTL
> (ASCP)
>
> On Sat, Jan 3, 2015 at 8:01 PM, Maria Mejia <mbmphoto <@t> gmail.com> wrote:
>
> > First, the very best of holidays to everyone.
> >
> > Now for the histology part. Our lab's focus is on the early stages of
> > Alzheimer's Disease in the Brainstem
> > using celloidin processing & embedding for IHC staining. This year, our
> > lab will be receiving 6 post-mortem
> > whole human brains (1 every other month). After fixation, processing &
> > celloidin embedding, the whole brain
> > will be serially cut at 100um thick. Each brain section will be 5 inches
> > x 4.5 inches in size.
> >
> > I will given 250 of these whole brain sections to stain for tau
> > IHC...that's 1500 whole brain sections/year!!!
> > 1) Does anyone have experience doing manual IHC staining of large
> > free-floating brain sections?
> > 2) What type of staining tools, dishes or other essential equipment can
> > anyone recommend?
> > 3) What's the most efficient way to stain 250 sections for batch IHC
> > staining - such as transferring batch
> > sections (maybe 5-10) from reagent to reagent?
> > 4) What type of batch apparatus to use?
> >
> > As for the antibody & ABC steps, I was thinking of placing each section
> > inside a large glass cigar tube
> > (yep, people use large glass tubes with fitted cap to store cigars), with
> > 5ml of antibody or ABC reagent & gently agitate on
> > a shaker/rotator at room temp during the incubation. Does anyone have
> > ideas on this?
> >
> > Please, any ideas, suggestions or recommendation anyone can provide will
> > be most greatly appreciated.
> >
> > Best regards
> > Maria Mejia
> > UCSF
> > Department of Neurology
> > San Francisco, CA
> >
> >
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> >
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