11/14/97

would appreciate any methods or references

TIA Joan Clark

j.clark@zoology.unimelb.edu.au

I, too, use the Paragon stain, but I do get a polychrome effect.

I was wondering if you use a saturated Sodium Borate solution to enhance

your staining. After drying the section on the hotplate, I put a 3 to 4

drops of Paragon stain and one drop of sodium borate on the slide (still

on the hotplate). The combination of heat and sodium borate enhance the

Paragon stain quite nicely. You might want to try this before adding H

& E to your "to do" list.

Sharron G. Chism HT (ASCP)

Electron Micropscopy Lab

Harris Methodist Fort Worth

Fort Worth, Texas

SharronChism@hmhs.com

epoxy semi-thin sections, but it would be very nice if we had some kind

of rapid H&E like stain that could give that kind of polychrome

differentiation between the nucleus and the cytoplasm.

The Paragon stain is supposed to be a polychrome stain, but what we in

effect see is more of a monochrome direct stain.

Does anyone know of a quick yet superior stain that might give us better

results here?

GBurgess@exchange.hsc.mb.ca

Eosin after removing the plastic. A stain like this is almost indistinguishable

from H&E and in fact microtomists may all be using Celestin blue B as the

supplies of tropical logwood (source of dye for hematoxylin) dry up.

Robert Mixon

mixonr@ohsu.edu

I need, and I'm sure it would be usefulto the rest of the list if it's as

wonderful as it sounds. TIA.

Lesley Weston.

lesley@unixg.ubc.ca

The resins that you mentioned (Spurr, Epon, and Araldite), all need to be

removed (etched) prior to staining with either aquous or alcoholic based

dyes(there are exceptions).

These resins can be etched (removed) with a solution called "Ethoxide".

Ethoxide is made as follows:

Make a saturated solution of Sodium Hydroxide in 100% Ethanol and let stand in

the dark for at least 48 hours. The solution will turn yellowish when ready.

To make this into a working solution mix equal parts of the above solution and

tolulene.

In a fume hood wearing DOUBLE gloves, eye protection, etc. dip the slide gently

in the working solution untill you can see that the resin has been removed, dip

the slide in 95% ethanol for a minute and then place in a buffer (pH 7.4) for at

least 5 minutes prior to staining. You can stain with your normal procedure for

the H&E starting with a DI water rinse. I have even done some beautiful

Trichromes on sections treated this way.

Some things to be mindful of though........

Do only one slide at a time as it it is very easy to etch out the tissue too.

These sections are much thinner in general than the paraffin sections normally

stained with H&E and you may have to increase the staining times a little.

Ethoxide is VERY caustic!!! One drop can do you a lot of damage and pain. I

lost a fingernail the second time I used the stuff because of a hole in my

glove, it was one of the most painful experiences I've ever had!

There may be a safer and/or easier way to do this but this was what I did before

GMA hit the market place back in the early 70's (almost in my 5th decade ;-)).

I also have to admit that I haven't done any plastic work in a few years so this

could very well be out of date(like me).

I'm going to cross post this to both the Microscopy and the Histology listservs

and hope that someone may have a better technique.

rschoonh@sph.unc.edu

Robert Schoonhoven

examine many

different vertebrate tissues (brain, skin, liver, kidney, etc)

embedded in

plastic resin (Eponate or LR White). We currently do a lot of LM

viewing of

2 micron sections, using toluidine blue as a counterstain. Some

tissues

have been fixed with aldehydes and osmium, for subsequent EM thin

section

studies. For immunocytochemistry, other tissues are only lightly

fixed with

aldehydes.

Our question:

Can you suggest other useful counterstains, besides

toluidine blue,

that are compatible with plastic sections. If you have a

favorite recipe,

please send to us off-line, so that we don't clog the listserver.

We'll

compile a list of the answers we receive, for those who are

interested.

Thanks in advance

David H. Hall hall@aecom.yu.edu

Dept of Neuroscience

Albert Einstein Col Medicine, Bronx, NY 10461

(718) 430-2195

(718) 430-8821 FAX

variation on the

hematoxylin theme for our epon sections. We use Heidenhain

hematoxylin-iron

which comes in two solutions (I have the recipes if you want to

make your

own) - an iron alum solution and a hematoxylin solution. Slides

are

preheated and stained on a hot plate that is maintained between

80 and 90

degrees C. Coat the sections with the iron alum solution for 2-10

min

depending on the tissue type, thickness, etc. Rinse VERY WELL

with distilled

water. Repeat the process on the hot plate with the hematoxylin

staining

solution using the same time interval as the iron alum. Because

the

"staining" only appears at this step, you may need to test a few

slides to

determine ideal timing. After rinsing well again with distilled

water, flood

the slides on the hot plate with TAP water to differentiate and

let sit for

3 min. Rinse briefly in distilled water and dry. Prestaining like

this

allows the slides to then go through autoradiography without

interfering

with the emulsion and has worked for us irregardless of the

fixative used.

Pat Hales

McGill University

Dept. of Anatomy & Cell Biology

hales@hippo.medcor.mcgill.ca

sections of vertebrate tissues embedded in plastic; our original standard

stain was toluidine blue.

We received 14 responses to this request, which we have edited to a ~7 page

file. Responses included stains such as hematoxalin, alcian blue, eosin,

Mayer's mucicarmine, methyl green, methylene blue/azure II, basic fuschin,

and Stevenel's blue. A copy of the file is available upon request by

e-mail. We have begun using a polychrome stain (see Van Reempts and

Borgers, 1975, Stain Tech. 50:19-23) with pleasing results.

Christine Roy and David Hall

Albert Einstein College of Medicine

Bronx, NY 10461

In the course of our work, we examine many

different vertebrate tissues (brain, skin, liver, kidney, etc) embedded in

plastic resin (Eponate or LR White). We currently do a lot of LM viewing of

2 micron sections, using toluidine blue as a counterstain. Some tissues

have been fixed with aldehydes and osmium, for subsequent EM thin section

studies. For immunocytochemistry, other tissues are only lightly fixed with

aldehydes.

Can you suggest other useful counterstains, besides toluidine blue,

that are compatible with plastic sections. If you have a favorite recipe,

please send to us off-line, so that we don't clog the listserver. We'll

compile a list of the answers we receive, for those who are interested.

Response I

If you etch your sections with EtOH/KOH or MeOH/KOH, you can do iron

hematoxylin, alcian blue, eosin and Mayer's mucicarmine, at least. PAS and

Feulgen *may* work even with un-etched epoxies--in theory, the hydrophobic

epoxy doesn't infiltrate the hydrophilic sites like DNA and polysaccharides

well, and so there's more access for aqueous solutions, even after

embedding (this probably explains much of the staining pattern you get with

Tol Blue O).

Whether or not the results of PAS or Feulgen ought to be trusted after glut

and osmium is a different question, however.... ;-)

Much of this is documented in an old paper by me and Seth Tyler:

Smith, J. and S. Tyler. 1984. Serial sectioning and staining of

resin-embedded material for light microscopy: recommended techniques for

micrometazoans. Mikroskopie 41:259-270.

Julian P.S. Smith III

Biology

Winthrop University

Rock Hill, SC 29733

803-323-2111 x227 (vox)

803-323-2246 (fax)

smithj@winthrop.edu

Response II

Methylene blue-azure II followed by basic fuchsin as a metachromatic stain

for animal

and/or plant tissues.

Bruce L. Wagner

Bessey Microscopy Facility

Iowa State University

blwagner@iastate.edu

Response III

I did a test of my own last year on 1 micron sections of mammalian tissue in

Spurr's resin. My goal was to find a very aggressive stain that would

provide contrast even to 100-200nm "thin" sections of cultured cell

monolayers for subsequent image analysis. I was successful! I tried

various stains recommended in Histology texts and commercial resources and

compared them to the standard 0.1% Toluidine blue in 0.1% sodium borate.

The ones which worked best were:

Azur II+ = Commercially available Azur II powder from EMS, made up at 1%

aqueous, with the addition of 0.1% borax.

ETS+ = 1 part commercially available Epoxy Tissue Stain from EMS plus 1 part

standard Tol. Blue in Borax.

Zymed's Methyl Green also worked well, about the same intensity as standard

Tol. Blue.

Note, subsequently I have used the above on LR White sections and they look

just as good, if not better, than the Spurr's.

Also, pretreatment with sodium metaperiodate as for immunolabeling seemed on

one occasion to result in darker staining (of 200 nm sections of cultured

cell monolayers) but on 1 micron sections the difference was not noticeable.

The other stains I tried (which mostly were too weakly staining to be

useful) were:

Epoxy Tissue Stain (commer. avail. from EMS, a mixture of Basic Fuchsin and

Tol. Blue)

Acid Fuchsin (1%) + Tol. Blue in Acetate or Borax

Thionin (0.1%) + Acridine Orange (1%)

Mayer's Hematoxylin (commer. avail. from Zymed)

Nuclear Fast Red (commer. avail. from Zymed)

Methylene Blue (commer. avail. from Zymed)

Azur II (commer. avail. from EMS, a mixture of Azur B and Methylene Blue)

Unpurified Methyl Green (0.2%)

Also tried pretreatment with sulphuric acid (supposed to increase stain

penetration) but it just took the sections right off the slides.

Karen Zaruba

kszaruba@mmm.com

Life Sciences Sector Lab

3M Company

3M Center 270-1S-01

St. Paul, MN 55144-1000

These opinions are my own and may not represent those of 3M.

Response IV

Methylene Blue/Azure II for EM thick sections and frozen sections. It

works on both epon and LR White. Shorter staining time for LR White.

Solution A: 1 gram Sodium Borate dissolved in 100 ml. H2O. Then add 1

gram methylene blue. Store in glass bottle.

Solution B: 1 gram Azure II dissolved in 100 ml. H2O.

Mix equal parts of Solution A and B. Filter before use. Staining time

10-60 sec.

Lee Dickey

LeeDMLM@aol.com

Response V

Toluidine Blue and Basic Fuschin for epoxy sections.

Request protocol from:

Lou Ann Miller

Univ. of Illinois

lamiller@uiuc.edu

Center for Microscopy & Imaging Home Page:

http://www.cvm.uiuc.edu/MicImagLab/MicImagLab.html

Response VI

Hot Carbol fuschin works in epon

Methylene blue, and methyl green in epon did not work well.

Rachel Teitelbaum

teitelba@aecom.yu.edu

Response VII

Stevenel's Blue is a reliable, intense stain for Spurr's one micron

sections.

The stain is quite stable.

The method, as adapted for plastic-embedded tissues can be found in:

Microscopa Acta 83:117-121 (1980).

del Cerro M, Cogen J, & del Cerro C;

Stevenel's Blue, an excellent stain for optical microscopical study of

plastic embedded sections.

You may also find interesting an article by R.W.Horobin,

Staining plastic sections: a review of problems, explanations and possible

solutions

J. Microscopy 131:173-186 (1983)

Mike Nesson, Ph.D.

Department of Biochemistry & Biophysics

2011 Ag&LS, Oregon State University, Corvallis, OR 97331-7305

(541)737-1866

FAX:(541)737-0481

nessonm@ucs.orst.edu

Response VIII

Tolivia, J.; Navarro, A.; Tolivia, D. (1994): Polychromatic staining of

epoxy semithin sections: a new and simple method. Histochemistry 101:

51-55.

Hans-Martin Vaihinger

Ruhr-University of Bochum

Comparative Endocrinology Research Section

Building ND 5/37

44780 Bochum

GERMANY

*********************************************************

phone ++49 234 700 4329

fax ++49 234 709 4551

e-mail Hans-Martin.Vaihinger@RZ.Ruhr-Uni-Bochum.de

Response IX

Variation on the hematoxylin theme for our epon sections.

We use Heidenhain hematoxylin-iron which comes in two solutions (I have the

recipes if you want to make your own) - an iron alum solution and a

hematoxylin solution. Slides are

preheated and stained on a hot plate that is maintained between 80 and 90

degrees C. Coat the sections with the iron alum solution for 2-10 min

depending on the tissue type, thickness, etc. Rinse VERY WELL with

distilled water. Repeat the process on the hot plate with the hematoxylin

staining solution using the same time interval as the iron alum. Because the

"staining" only appears at this step, you may need to test a few slides to

determine ideal timing. After rinsing well again with distilled water,

flood the slides on the hot plate with TAP water to differentiate and let

sit for 3 min. Rinse briefly in distilled water and dry. Prestaining like

this allows the slides to then go through autoradiography without interfering

with the emulsion and has worked for us irregardless of the fixative used.

Pat Hales

McGill University

Dept. of Anatomy & Cell Biology

hales@hippo.medcor.mcgill.ca

Response X

Jos van Rempts and Marcel Borgers:

A simple polychrome stain for conventionally fixed epon-embedded tissues.

Stain Technology 50, No 1, p.19, (1975).

I think that once you will try it you will always use it because staining is

spectacular, although takes a little bit of your time.

Wis Jablonski, OiC EM/X-ray Microanalysis, CSL, Uni of Tasmania

W.Jablonski@csl.utas.edu.au

Response XI

1. Electron Microscopy Sciences Catalog XII, p.31, Technical Tip

Superior Methylene blue-Azure II stain for semithin sections

2. Theory and Practice of Histotechnology, chp 19-Electron Microscopy (Nan

Pillsbury), section on staining thick sections p.342, lists 2% Trypan blue

in 1% sodium borate used

as is toluidine blue with pre-test for optimal time on Epon-embedded sections

3. I have found Haematoxylin & Eosin as is commonly used in histochemical

staining to work quite well (as does #1 above).

Winston W. Wiggins,

wwiggins@mail.carolinas.org

Supervisor, Electron Microscopy Lab

Carolinas HealthCare System

Charlotte, NC

USA

Response XII

Polychrome stain

1. Blue Stain: Methylene blue .13gm , Azure II .02gm, Glycerol 10ml,

Methyl alcohol 10ml, D.H2O 80ml

(stir and filter, keeps 6 mo.)

2. Red stain: STOCK SOL: Basic fuchsin .2gm, DH2O 100ml (stir and

filter, keeps 6mo.)

WORKING SOL: dilute stock 1:4 in DH2O (fresh daily)

3. Sodium Hydroxide 1% fresh daily

Procedure:

1. Flood slide with blue stain 15-60 seconds, depending on

temperature and material.

2. Add 4-6 drops NaOH to the stain and mix by tilting the slide,

about 10 seconds total time.

3. Wash in running water and dry on hotplate. Blue stain can be

destained by heating.

4. Add red stain for 15-30 seconds on hotplate. (Red stain cannot

be destained)

5. Rinse with running water and dry.

References: Mackay and Mead. 1970. 28th EMSA meetings pp.296-297.

Modified by Griffin and Fahrenbach, Oregon Regional Primate Research

Center

Linda M. Fox

Dept. of CBN and Anatomy

Loyola University Medical School

2160 S. First Ave.

Maywood, Illinois 60153

lfox1@wpo.it.luc.edu

Response XIII

Methylene blue-basic fuchsin stains described in handout

provided by LKB that compared these references and showed colored

micrographs.

References as follows:

Aparicio, SR, and Marsden, P: Methylene blue-basic fuchsin. Journal of

Microsc. (Eng.) 89:139-141, 1969.

Huber, JD, Parker, F, and Odland, GF; Basic fuchsin and alkalinized

methylene blue. Stain Technol. 43:83-87, 1968.

Humphrey, CD, and Pittman, Fe: Methylene blue-azure II and basic fuchsin.

Stain Technol 42:9-14, 1974.

Nancy A. Monteiro-Riviere, Ph.D.,BCFE,BCFM

Professor of Investigative Dermatology/Toxicology

North Carolina State University

College of Veterinary Medicine

Cutaneous Pharmacology and Toxicology Center

4700 Hillsborough Street

Raleigh, NC 27606

Telephone: 919-829-4426

FAX: 919-829-4358

email: Nancy_Monteiro@ncsu.edu

CTPC Homepage: http://cptc.ncsu.edu

Response XIV

LKB handbook

Title: Stains for Plastic Embedded Tissue Sections.

1. Comparison of three different methylene blue-basic fuchsin

stains.(Aparicio, Huber, Jha.) August 1977

2. Staining of sections from different animal, human, and plant tissues

with a methylene blue-azure ll-basic fuchsin stain.(Humphrey) April

1977.

Vijay H Bandu

bandu@emu.unp.ac.za

Since March, we have tried the reference given in response X. Osmicated

epon sections and non-osmicated LR White sections were stained with good

results. I increased the staining time from 15 minutes to 25 minutes in

celestine blue B solution (@ 60 C). I also increased the staining time in

Modified Cason solution from 10 minutes to 20 minutes (@ room temp.) The

increase in staining times resulted in better staining. I also found it

necessary to rinse in several changes of 0.1 N HCl and dH20 to completely

rinse off the stain before drying.

Thank you to all those who contributed.

Christine Roy

David H. Hall, PhD

David Hall

Department of Neuroscience

Albert Einstein College of Medicine

Bronx, NY 10461 phone (718) 430-2195 FAX (718) 430-8821