[Histonet] HELP! Need some old fashioned histology advice

Jay Lundgren jaylundgren <@t> gmail.com
Tue Jan 6 16:26:58 CST 2015 

     I don't know if the slides were subbed or not, but they looked
improvised.  The point is, at some point the sections are going to have to
be mounted for visualisation, right?  Someone or something is going to look
at the section under magnification?

     If you mount them on glass *before* staining, the whole problem of
batching becomes one of improvising a giant stain rack and giant staining
line.  Don't forget, you need giant cover slips, giant slide folders, and
giant postdocs.
I know that lab glassware is custom ordered every day.  Sounds expensive
and time consuming to set up, but it beats fiddling around with free
floating tissue anyday, IMHO.

    I don't know what the Ab penetration is going to be like on a 100um
section, because they weren't using IHC at Genentech, they were doing in
vitro DNA hybridization on the slides.  I'm assuming the section thickness
is necessary because the PI wants to follow axonal pathways and see the
patterns of staining?  Sounds like a fun project.  If you need more help
later you can PM me.

                                               Sincerely,

                                                      Jay A. Lundgren,
M.S., HTL (ASCP)

On Mon, Jan 5, 2015 at 10:38 PM, Mejia, Mary <Mary.Mejia <@t> ucsf.edu> wrote:

>  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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