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
prueg...@hotmail.com
Date: Sun, 4 Jan 2015 11:58:38 -0600
From: jaylundg...@gmail.com
To: mbmph...@gmail.com
Subject: Re: [Histonet] HELP! Need some old fashioned histology advice
CC: histonet@lists.utsouthwestern.edu; mary.me...@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 mbmph...@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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