[Histonet] Dewax for IF

From:"Gudrun Lang"

 Melissa,
We dewax like this: 3x5 min xylene, 100-96-80-50-A.d. each 1 min
We do IF on FFPE renal biopsies.
Greetings Gudrun

-----UrsprŘngliche Nachricht-----
Von: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] Im Auftrag von Melissa
Mazan
Gesendet: Montag, 24. Juli 2006 19:23
An: histonet@lists.utsouthwestern.edu
Betreff: [Histonet] Re: Histonet Digest, Vol 32, Issue 24

Hi all,
Can those of you who do non-automated dewaxing share their protocols with
me? At first I was doing 3X 3 minutes xylene, then 1X 2 minutes 100% EtOH,
then one minute of 85%, and one minute of 75%.  Having problems with antigen
labeling (immunofluorescence) so am worried that it might be my dewaxing
protocol.  Also, how often do you completely change out the xylene? Thanks
in advance - Melissa

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> Today's Topics:
> 
>    1. Gomori's Trichrome on Cementum of a Human Tooth (Ho, Sunita)
>    2. Rapid Tissue Processor (a.schmidt@fuse.net)
>    3. Re: Fluorophore adsorption emission and spectral separation
>       (Adam Perry)
>    4. RE: Commercial Decalcifying Solns. (patsy ruegg)
>    5. IHC validation of blocks and anitbodies (Jessica Piche)
>    6. CAE and Mature Mast cells look at procedure (Madary, Joseph)
>    7. Postfixation of frozen brain tissue (Robert J. Schloesser)
>    8. Can Alk phs be run on peroxidase slides? (Dana Settembre)
> 
> 
> ----------------------------------------------------------------------
> 
> Message: 1
> Date: Sun, 23 Jul 2006 13:54:35 -0700
> From: "Ho, Sunita" 
> Subject: [Histonet] Gomori's Trichrome on Cementum of a Human Tooth
> To: histonet@lists.utsouthwestern.edu
> Message-ID:
> 	<0D6FC3C553E12A43B7C6B1F4A9DA429301F60D8B@EXVS05.net.ucsf.edu>
> Content-Type: text/plain; charset=us-ascii
> 
> Hi All,
> 
> I recently stained a decalcified human cementum tissue with Gomori's 
> Trichrome (using a standard procedure provided in any histology text 
> book).  I have regions of red and blue - Cementum was predominantly 
> stained red and dentin blue (consistently observed between many 
> specimens).  I am assuming that this was primarily due to packing 
> density of collagen, more in cementum than in dentin.  The cementum 
> around the lacunae was stained blue.  Is my assumption correct or is 
> there a better technical answer for what I observed using a light 
> microscope?  I look forward to your thoughts.
> 
> Thanks in advance.
> 
> Sunita
> 
> 
> 
> 
> ------------------------------
> 
> Message: 2
> Date: Sun, 23 Jul 2006 19:46:32 -0400
> From: 
> Subject: [Histonet] Rapid Tissue Processor
> To: Histonet@lists.utsouthwestern.edu
> Message-ID: <22333582.1153698392694.JavaMail.root@webmail6>
> Content-Type: text/plain; charset=utf-8
> 
> We have been using the Sakura Rapid Tissue processor for about 3 months
now. We have been having trouble with our digested PAS stain on liver needle
biopsies using the Ventana Nexes stainer. Has any other facility experinced
any problems with their special stains or IHC stains and if you have this
processor I would appreciate hearing any of your feelings about it. Tahkns
for the input.
>                                                   Angie Schmidt
>                                                     Tri-Health 
> Labs--Cincinnati
> 
> 
> 
> ------------------------------
> 
> Message: 3
> Date: Mon, 24 Jul 2006 07:47:45 -0700 (PDT)
> From: Adam Perry 
> Subject: Re: [Histonet] Fluorophore adsorption emission and spectral
> 	separation
> To: Histonet@lists.utsouthwestern.edu
> Message-ID: <20060724144745.41330.qmail@web30401.mail.mud.yahoo.com>
> Content-Type: text/plain; charset=iso-8859-1
> 
> I had an immunofluorescence question that I'd love to get some input on
from anyone out there.  I'm trying to colocalize two antigens in rodent
brain sections.  One of the antigens is yielding fairly weak staining- it
also happens to be the one that I'm visualizing with Alexa Fluor 488, so I'm
getting fairly high background autofluorescence- and I think that the
staining could be improved by reducing the autofluorescence.  My DAPI
staining (ex/em: 350/470) is great...it only stains nuclei with minimal
autofluorescence.  They offer Alexa Fluor 405 streptavidin which has a
similar excit/emission range, so I thought labeling with the Alexa Fluor 405
would produce a similarly "clean" background as my DAPI labeling.  Also, I
already have the filter sets to work with Alexa Fluor 405 (as opposed to
going to the other end of the spectrum).  
>    
>   Has anyone used Alexa Fluor 405?  What is your impression of tissue
autofluorescence in that range, photostability/bleaching of the compounds,
comparison of signal using other fluorophores, etc.
>    
>   Any input would be greatly appreciated.
>   Thanks,
>   Adam Perry
>    
>   Department of Neuroscience
>   University of Illinois at Chicago
>   Chicago, IL 60612
>   
> 
> Gayle Callis  wrote:
>   Moran,
> 
> Molecular Probes, go to resources on home page for MP, and click on 
> their spectraimager. You can play with the fluorophores you need to 
> see (do it as antibodies) and see the graphs for separation - for both 
> CLSM and fluorescent microscope work.
> 
> With our confocal, adjustment can be made to eliminate 
> autofluorescence. I also have a review article on autofluorescence, 
> written with GFP in mind, but useful for immunofluorescent work also.
> 
> If you want the review, I will send via private email.
> 
> 
> Gayle Callis
> MT,HT,HTL(ASCP)
> Research Histopathology Supervisor
> Veterinary Molecular Biology
> Montana State University - Bozeman
> PO Box 173610
> Bozeman MT 59717-3610
> 406 994-6367
> 406 994-4303 (FAX)
> 
> 
> 
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> 
>  		
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>  Get on board. You're invited to try the new Yahoo! Mail Beta.
> 
> ------------------------------
> 
> Message: 4
> Date: Mon, 24 Jul 2006 09:19:49 -0600
> From: "patsy ruegg" 
> Subject: RE: [Histonet] Commercial Decalcifying Solns.
> To: "'Marilyn Johnson'" ,
> 	
> Message-ID: <200607241519.k6OFJhlG059684@pro12.abac.com>
> Content-Type: text/plain;	charset="US-ASCII"
> 
> I prefer Immunocal by Decal Chemicals, it seems to be just as fast as 
> the more harsh HCL based decals like RDO but much much better for IHC, 
> it is formic acid based.
> Patsy
> 
> Patsy Ruegg, HT(ASCP)QIHC
> IHCtech
> 12635 Montview Blvd. #216
> Aurora, CO 80010
> 720-859-4060
> fax 720-859-4110
> pruegg@ihctech.net
> www.ihctech.net
> -----Original Message-----
> From: histonet-bounces@lists.utsouthwestern.edu
> [mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of 
> Marilyn Johnson
> Sent: Saturday, July 22, 2006 8:47 AM
> To: histonet@lists.utsouthwestern.edu
> Subject: [Histonet] Commercial Decalcifying Solns.
> 
> Hi Histonetters,
> Could anyone provide me with their preferences for commercially 
> prepared decalcifying solns?
> These solns will be required for routine bone sections for H&E 
> staining and not biopsies or rush specimens.
> Please provide company names and order numbers.
> Thank you in advance for any replies.
> 
> Marilyn Johnson
> Alberta Agriculture
> Edmonton, AB. Canada.
> _______________________________________________
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> 
> 
> 
> 
> ------------------------------
> 
> Message: 5
> Date: Mon, 24 Jul 2006 09:17:45 -0700 (PDT)
> From: Jessica Piche 
> Subject: [Histonet] IHC validation of blocks and anitbodies
> To: histonet 
> Message-ID: <20060724161745.56186.qmail@web82011.mail.mud.yahoo.com>
> Content-Type: text/plain; charset=iso-8859-1
> 
> Hi All,
>    
>   I have a few questions regarding IHC. When validating controls and new
antibodies do you always run a negative control slide as well? Can anyone
share their protocols with me on validating controls, and new antibodies?
Any help would be appreciated. Thank you.
>    
>   Jessica Piche-Grocki, HT (ASCP)
> 
> 
> ------------------------------
> 
> Message: 6
> Date: Mon, 24 Jul 2006 12:22:19 -0400
> From: "Madary, Joseph" 
> Subject: [Histonet] CAE and Mature Mast cells look at procedure
> To: 
> Message-ID:
> 	<7CAB706201F11843BD26AD516326F0C8010D3890@MD1MS007.medimmune.com>
> Content-Type: text/plain;	charset="iso-8859-1"
> 
> Okay my histoheroes and heroines,
> One of the investigators here asked for a CAE designed for plastics(but
supposedly works on FFPE).  I did this protocol, but it isn't working. I am
having a rough time because I am not used to making mistakes(kidding).  She
needs mature mast cells only, I gave her 7 different Mast cell procedures
that all worked very well(even the dominicis), and one Alcian Blue Safranin
that stains Heparin containing Mast cells red Amine containing Mast cells
blue. So 2 questions are:
> 
> 1-Do mature cells stain differently?
> 2-Does anyone have a decent CAE they can share?  Here is the one I used
and the control is fresh rat skin and all the other procedures worked on
that block. 
> 
> 	Chloroacetate Esterase (CAE) Staining
> 
> Preparation of Solutions
> 
> 1.	New Fuchsin Solution
> 			New Fuchsin				1g
> 			2N hydrochloric acid (HCl)	          25ml
> 
> 2.	4% Sodium Nitrite Solution
> 			Sodium Nitrite				1g
> 			ddH2O
25ml
> 
> 3.	Phosphate Buffer (0.1M, pH 7.6)
> 		Stock Solutions
> A.	0.2M solution of monobasic soduim phosphate (27.8g NaH2PO4 in 1L
H2O)
> B.	0.2M solution of dibasic sodium phosphate (28.4g Na2HPO4 in 1L H2O)
>       --> To make pH 7.6 Solution: mix 6.5mL of A, 43.5mL of B, and 50mL
of ddH2O.
> 
> 4.	Naphthol AS-D Chloroacetate Solution (Store at -20░C)
> 		Naphthol AS-D Chloroacetate		10mg
100mg
> 		N-N Dimethyl Formamide		              5ml
50ml
> 
> 
> Staining Solution (make fresh before each use)
> 
> In a single vial:	1) mix New Fuchsin and 4% Soduim Nitrite
> 			2) add phosphate buffer and mix
> 			3) add Naphth AS-D Chloroacetate Soultion and mix
well
> 
> 	Use 1 ml freshly mixed solution for approx 4 tissue slides
> New Fuchsin				2.5Ál		5Ál
12.5Ál		25Ál
> 4% Sodium Nitrite			2.5Ál		5Ál
12.5Ál              25Ál
> Phosphate Buffer	                           1ml               2ml
5ml             10ml
> Naphthol AS-D Chloroacetate	  50Ál	         100Ál		  250Ál
500Ál
> 
> Staining Procedure
> 1.	Stain with fresh staining solution for 20 min. 
> 2.	Wash with running tap water (For plastic sections use wet cotton
swab wipe off dusty residue).
> 3.	Counterstain with Gill's II hematoxylin for 20 to 45 seconds (or
methyl green for 30 seconds).
> 4.	Wash with warm tap water to blue  (longer will result darker blue,
or dip into 50% of lithium carbonate for 5 - 20 seconds for deeper blue,
then rinse with water).
> Dry and mount with mounting medium. (For paraffin sections, quick
dehydration before mounting).
> 
> 
> 
> ------------------------------
> 
> Message: 7
> Date: Mon, 24 Jul 2006 12:41:50 -0400
> From: "Robert J. Schloesser" 
> Subject: [Histonet] Postfixation of frozen brain tissue
> To: 
> Message-ID: <000001c6af40$0ff4ff80$76e4bb89@yourd54swmkxh6>
> Content-Type: text/plain;	charset="iso-8859-1"
> 
> Hi All,
> 
> We have to do IHC on fresh frozen brain samples perfused only with 
> saline. I am only used to cutting and IHC on PFA fixed brains.
> Therefore I have many questions on how to treat fresh frozen samples. 
> 
> Postfixation on slides: PFA or Acetone/Ethanol? 
> Is there a reliable way to postfix whole blocks?
> Is there a way to postfix 30-200Ám thick slides without mounting them 
> on slide (to do free floating staingin)?
> 
> Any kind of input, protocols or tips&tricks and do`s and don`t are 
> highly appreciated.
> 
> Thank you very much
> 
> Robert
>  
> 
> 
> 
> ------------------------------
> 
> Message: 8
> Date: Mon, 24 Jul 2006 12:44:48 -0400
> From: "Dana Settembre" 
> Subject: [Histonet] Can Alk phs be run on peroxidase slides?
> To: 
> Message-ID: 
> Content-Type: text/plain; charset=US-ASCII
> 
> Hi,
> One of my pathologists is asking if I can take a slide that has 
> already been stained with a peroxidase/avidin/biotin/DAB detection 
> system and re-stain with an Alkaline Phosphotase detection system.
> Can it work?
> Must it be de-stained?
> 
> Any info will help.  Thanks,
> 
> Dana Settembre
> Immunohistochemistry Lab
> University Hospital - UMDNJ
> Newark, NJ
> 
> 
> 
> ------------------------------
> 
> _______________________________________________
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> Histonet@lists.utsouthwestern.edu
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> 
> End of Histonet Digest, Vol 32, Issue 24
> ****************************************

--
Melissa R. Mazan, DVM, Diplomate ACVIM
Associate Professor of Large Animal Medicine Director of Sports Medicine
Cummings School of Veterinary Medicine Tufts University 200 Westborough Road
North Grafton, MA 01536
Tel: 508-839-5395
Fax: 508-839-7922
Email: melissa.mazan@tufts.edu


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