Fixation, Embedding, Cutting, Slide, Order of stains
(Company and Catalogue Nb (german version))
Fixation
Neutral formalin
4% (10%) phosphate buffered formalin (0.1M, pH 7.4), without any additives and not
recycled.
Duration of fixation
Needle biopsy: 4 - 12 hours
Wedge biopsy: 12 hours
NOTE: Formalin is the best-suited (only) fixative for immunohistochemical studies on
paraffin embedded tissue. EM studies are also possible after formalin fixation.
To avoid deleterious effects of technical grade or contaminated formalin on
immunohistochemistry 4% paraformaldehyde in 0.1 M phosphate buffer (ph 7.4) is
recommended (http://www.ihcworld.com/protocols/histology/fixatives.htm).
Bouin
15 parts saturated picric acid, aqueous (15 g picric acid (Fluka 74069,
www.sigmaaldrich.com) + 950 ml Aqua dest.)
5 parts formalin 37% (Merck Art. 1.04003, www.merck.de)
1 part glacial acetic
NOTE: Bouin may be used for post fixation for special stains (e.g. AFOG-stain)
Embedding
Paraffin (Paramat)
NOTE: To preserve antigenicity for immunohistochemical studies avoid heating
above 600C during embedding.
Rapid embedding
Laboratories receiving transplant biopsies on a regular basis should have a rapid
embedding procedure available, so that the clinicians receive the biopsy results
within 4-6 hours.
NOTE: We reduce all steps in the tissue processing to 10 min. each. Others run all
biopsies
on automated processors in 4 -5 hours.
Section thickness and cutting
2 -3 µm section thickness, for cutting renal biopsies a rotating microtome should be
used.
For the Congo red von Kossa and Alizarin red S stain thick sections are necessary
((4)-6-8µm) (see below).
Section thickness should always be the same. Per glass slide 2-3 sections should be
mounted, so that a total of 25 - 40 sections are available for the evaluation.
NOTE: We recommend the following procedure: The sections should be in sequence
and the slides numbered so that it is possible to follow one lesion through the whole
series of sections. We also recommend to cut 3 sections (unstained) for potential
additional use/studies. This avoids re-cutting and tissue loss when adjusting the
paraffin blocks on the microtome.
Type of slide
Superfrost untreated (www.menzel.de)
Special slides are only used for:
AFOG stain (polylysine coated slides)
Immunohistochemistry: Superfrost Plus
Immunofluorescence: Superfrost Gold (very expensive); alternative see
under immunofluorescence
Mounting medium
Mount as usual
Order of stains
The order of stains should always be the same and is in our lab the following:
HE, Resorcin Fuchsin van Gieson, PAS, AFOG, PASM, PAS, HE, etc (see under
stains).
NOTE: The head technician should check the first and second HE for the number of
glomeruli. If less than 5 glomeruli are present, serial sections of the whole biopsy
should be performed immediately. Every second section is stained with PAS i.e. PAS
– unstained section - PAS- unstained section etc. More than five sections per slide
should be mounted in case serial sections are necessary.
Serial sections are also recommended in case of childhood nephrotic syndrome and
with less than 15 glomeruli in the initial series of sections
Control sections
For all special stains (PAS, AFOG, PASM etc.) Control sections with well-known
strong positivity should be used.
NOTE: The quality of diagnosis is the result of an optimal technique and a welltrained pathologist
Stains
Hematoxylin – Eosin (HE)
Staining procedure
1. Dewax and bring to water
2. Stain in Mayer's hematoxylin*
3. Rinse briefly in tap water
4. Differentiate in HCl-alcohol 0.5%
5. Rinse in tap water and blue
6. Erythrosin
7. Rinse briefly in tap water
8. Dehydrate through alcohols, clear in xylol
9. Mount sections as usual
8 min.
3x brief
10 min.
2 min.
Solutions
Mayer's hematoxylin:
Boil 2 g Hematoxylin crystalline in distilled water. (Merck Art. Nr. 1.04302,
www.merck.de)
Add to:
1000 ml Distilled water
Add:
0.2 g Sodium iodate (Merck Art. Nr. 1.06525, www.merck.de)
50 g Dissolve the potassium alum at room temperature (Aluminiumpotassium sulfate
Merck Art. Nr. 1.01042. www.merck.de)
20 g Chloral hydrate (Merck Art. Nr. 1.02425)
1 g Citric acid (to prevent precipitations) (Merck Art. Nr. 1.00244, www.merck.de)
Erythrosin:
Erythrosin A 1%
(REACTIFS RAL, France, /www.reactifs-ral.fr)
Alternative for Mayer' hematoxylin: Hematoxyline (Papanicolaou)
(J.T.Baker,Ref.3873, www.mallbaker.com)
Results
Nuclei
Connective tissue
Red blood cells
—
—
—
blue
pale pink
pink
PASM (Periodic Acid + Silver Methenamine)
Staining procedure
1. Dewax and bring sections to distilled water
2. Prepare fresh silver solution and pre-heat (in 60°C oven)for exactly 30 min.
3. Cool slides to room temperature
15 min.
4. Place sections in 1% periodic acid
15 min.
5. Flush sections 3 times with distilled water.
6. Stain with silver solution in oven at 60°C.
30-50 min.
7. Rinse in distilled water.
8. Gold chloride solution 0.25% until sections are grey
9. Rinse in distilled water.
10. Dip sections quickly three times into 3% sodium thiosulphate
11. Wash in tap water for 2 min.
12. Dehydrate through alcohols, clear in xylol
13. Mount as usual
Solutions
- Periodic acid 1% aqueous (Fluka 77310, www.sigmaaldrich.com)
- Sodium thiosulphate 3% aqueous (Merck Art. 1.06512, www.merck.de)
- Borax 2% aqueous (Merck Art. 1.06308, www.merck.de)
- Methenamine 3% aqueous (store in refrigerator)
(Merck Art. 1.04343, www.merck.de)
Silver nitrate 5% aqueous (Merck Art. 1.01512, www.merck.de)
Gold chloride 0.1% aqueous (pharmacy)
Silver working solution:
5 ml silver nitrate 5%
5 ml Borax solution 2%
40 ml Methenamine 3%
Results
Basement membranes of glomeruli and tubules deep black.
NOTE:
1. After staining for 30 min. in silver solution control sections under the
microscope, continue staining until the glomerular basement membranes are
deep black. Tubular basement membranes stain stronger, so concentrate on
the glomeruli when checking the stain under the microscope
2. All solutions must be prepared with distilled water.
3. Silver nitrate-solution must be prepared shortly before use.
4. Glassware must be cleaned for 1 hour in distilled water and emptied
immediately before use.
Weigert’s Resorcin - Fuchsin
(Elastic stain)
Staining procedure
1. Dewax and bring sections to 96% alcohol
2. Bring sections from 96% alcohol immediately in Resorcin-Fuchsin-solution
30 min.
4. Rinse sections in running tap water
5. Stain in Weigert's Iron Hematoxylin
3 min.
6. Rinse sections in running tap water
7. Differentiate briefly three times in HCl-alcohol 0.5%
8. Rinse sections in running tap water, blue
10 min.
9. Van Gieson solution
5 min.
10. Rinse briefly in running tap water
11. Dehydrate quickly through alcohols, clear in xylol
14. Mount as usual
Solutions
Ready for use:
100 ml Resorcin-Fuchsin-stain (Weigert, Chroma 2E 030 (= stock solution),
www.chroma.de)
100 ml HCl alcohol 0.5%
Weigert's iron hematoxylin:
Solution I:
10 g hematoxylin crystalline (Merck Art. 1.04302, www.merck.de) in
1000 ml alcohol 96%
Dissolve properly and store.
NOTE:
This solution must be stored for 1 month at room temperature in bright light.
Solution II:
40 ml ferric chloride (anhydrous) (Hänseler AG 21-5401-02, www.haenseler.ch)
10 ml HCl 25% (Merck Art. 1.09911, www.merck.de)
dissolve in :1000 ml distilled water.
This solution is immediately ready for use.
Mix solution I and solution II immediately before use at equal parts (v/v).
Van Gieson picric acid - acid fuchsin:
Solution I
Add 15 g picric acid to 950 ml distilled water Store for one week, shake and filter.
Solution II
Dissolve 2 g acid fuchsin (Acid fuchsin (Rubin S) Merck Art. Nr. 7629, www.merck.de)
in 50 ml distilled water and filter.
Mix solution I and solution II immediately before use at equal parts (v/v).
Results
Elastic membranes
Nuclei
Collagen
Other tissue elements
— deep black or bluish black
— blue to black
— red
— yellow
PAS Perjodic Acid Schiff
Staining procedure
1. Dewax and bring sections to distilled water
2. Transfer sections in periodic acid 0.5%
3. Wash with distilled water
4. Schiff's reagent
5. SO2-containing water (metabisulphite rinses)
6. Place in running tap water
7. Hematoxylin (nuclear stain)
8. Wash in running tap water, blue
9. Dehydrate through alcohols, clear in xylol
10. Mount as usual
5 min.
twice each
10 min.
3 min.
10 min.
2 min.
10 Min.
Solutions
Periodic acid 0.5% aqueous (Fluka 77310, www.sigmaaldrich.com)
Schiff s-reagent (pharmacy) at room temperature
SO2-containing water
10 ml 10% solution of water free sodium bisulphite (Merck Art. 1.06528
www.merck.de)
10 ml 2N HCl (pharmacy)
200 ml tap water
Mayer's hematoxylin or alternative: see HE stain
Results
Glycosaminglycanes
Basement membranes
Nuclei
— strong violet
— strong violet
— blue
Note:
1. Stain always test sections with known strong positivity.
2. Use only freshly prepared periodic acid.
3. Store Schiff's reagent in the refrigerator at 40 C. Warm before use to room
temperature.
Acid fuchsin orange G-stain (AFOG)
Slides: Polylysin coated slides preferred
Staining procedure
1. Dewax and bring sections to water
2. Post-fixation in Bouin's solution (for 2 hours in case of surgical biopsies or
nephrectomy specimens, needle biopsies for at least 1 hour) in oven at 60° C
and afterwards at room temperature up to 24 hours (needle biopsies 12
hours).
3. Wash in water until sections are white
20 min.
4. Stain nuclei with Weigert's iron hematoxylin
1 min.
5. Differentiate three times quickly in 1% HCl alcohol
6. Rinse in tap water
10 min.
7. Treat with aqueous 1% phosphomolybdic acid
5 min.
8. Rinse with distilled water
9. Staining solution (AFOG)
5 min. /10 min
10. Rinse quickly in water.
11. Dehydrate quickly through alcohols, clear in xylol
12. Mount as usual
Weigert's iron hematoxylin:
Solution I: 10 g hematoxylin crystalline (Merck Art. 1.04302, www.merck.de) in1000
ml alcohol 96%
Dissolve properly and store.
Note:
This solution must be stored for 1 month at room temperature in bright
light.
Solution II: 40 ml ferric chloride (anhydrous) (Hänseler AG 21-5401-02,
www.haenseler.ch)
10 ml HCl 25% (Merck Art. 1.09911, www.merck.de)
dissolve in :1000 ml distilled water.
This solution is immediately ready for use.
Solution I and II should be mixed properly immediately before use.
AFOG-solution:
Boil 5 g Water Blue (Fluka Art. Nr. 95290, www.sigmaaldrich.com) in 1000 ml distilled
water.
After cooling, add 10 g Orange G (Chroma Art. Nr. 1B 221, www.chroma.de) and
15 g acid fuchsin (Fuchsin S, Fuchsin Acid) (Chroma Art. Nr. 1B 525, www.chroma.de)
Adjust to pH 1.09 by adding HCl (25%; about 30 ml)).
Note:
The pH of the AFOG-solution is crucial for the staining results. Check pH always
before use.
Comment:
1. Postfixation of dewaxed sections in Bouin's solution is crucial for the staining
results. Without postfixation no optimal staining of protein deposits is possible! As a
rule of thumb: the longer the better!
Results
Basement membranes
— pale blue
Mesangial matrix
— blue
Cytoplasm of cells
— grey-yellow-orange
Nuclei
— (orange)-brownish!-black
Collagen
— blue
Red blood cells
— yellow-orange -brownish
Serum
— yellow to orange
Protein deposits in glomeruli, vessels and tubular BM — lively red (homogeneous)
Fibrin
— red-orange (fibrillar),
Amyloid
— pale bluish with a reddish centre
Sirius Red
Staining procedure
1. Dewax and bring sections to water
2. Place sections in celestin blue after gentle stirring with one slide 7 min.
3. Transfer sections to Mayer's hematoxylin
7 min.
4. Rinse briefly in water
5. Differentiate in HCl-alcohol 0.5% (until blue precipitate is washed away)
6. Blue
5 - 10 min.
7. Picro-Sirius red
30 min.
8. Wash briefly in water
9. Rapid dehydration ( 3 x abs. alcohol)
10. Xylol
11. Mount as usual (Pertex)
Solutions
Celestin blue:
Dissolve 40 g ferric ammonium sulphate over night in 800 ml distilled water
Add 4 g Celestin blue (Celestine blue B Chroma 1A 242 (www.chroma.de) or
Celestine Blue 206342 ALDRICH, www.sigmaaldrich.com)
Boil the mixture for 3 min. and filter.
Add 112 ml glycerin (Merck Art. 1.04093, www.merck.de)
Picro-Sirius red:
Sirius red 0.1% (F3 BA Chroma 1A 280, www.chroma.de) in saturated picric acid
Saturated aqueous picric acid:
Dissolve 15 g picric acid (Fluka 74069, www.sigmaaldrich.com) in 950 ml distilled
water
Store for a couple of days at room temperature. Picric acid must be “truly” saturated
(precipitate should be present!), stir before use.
Mayer's hematoxylin (see HE)
Results
Collagen
Nuclei
—
—
red
blue
NOTE
1. The exact concentration of Sirius red is important for optimal staining results.
2. The solution may be stored for years at room temperature
3. In case picric acid shows only a pale orange colour, the solution is not saturated,
add more picric acid until the colour is bright orange, store for 24 hours at room
temperature.
4. Celestin blue-solution cannot be stored indefinitely, as soon as the colour
becomes brownish, fresh Celestin blue solution must be used. Simple test for the
usability: One drop of Celestine blue on a piece of white cotton should give a bright
blue centre with a small brownish rim (Fig: see below). In case of a broad brownish
rim fresh solution must be prepared.
Congo Red
Note: use thick sections 6-8µm only! Stain sections for KMnO4 +
Congo red and Congo red (only) side by side. Mark one section
“KMnO4” and the other one “Congo”.
Staining procedure
1. Dewax and bring sections to distilled water
2. Place section “KMnO4” in KMno4 solution
(keep the “Congo” section in distilled water)
3. Rinse in distilled water
4. Oxalic acid (3%)
5. Rinse in distilled water
6. Place both sections in Congo red solution
7. Wash in tap water
8. Stain with hematoxylin
9. Wash in tap water
10. Differentiate briefly three times in HCl-alcohol 0.5%
11. Wash in tap water and blue
12. Dehydrate through alcohols, clear in xylol
13. Mount as usual (Pertex)
3 min.
3 min.
30 min.
1 min.
4 min.
10 min.
Solutions
Potassiumpermanganat:
15 gr KMnO4 (Merck Art. 1.05082 (www.merck.de)
300 ml distilled water
300 ml sulphuric acid (0.3% =0.9 ml sulphuric acid conc. (Merck Art. 1.0731)
dissolved in 300 ml distilled water)
Mix before use
Oxalic acid
3% aqueous (Merck Art. 1.00495)
Congo red:
500 ml alcohol (80%) saturated with 2.5 gr Congo red (Merck Art.1340)
Add 1 gr Potassium hydroxid (Merck Art. 5032. Sore over night before use, then
filtrate
Mayer's hematoxylin (see HE)
Results
All types of amyloid
— orange-red, green in polarized light
After KmnO4 pretreatment: AA-amyloid negative or slightly reddish, but no green
color after polarisation (KMnO4-sensitive amyloid), all other types of amyloid are
resistant against KMnO4 pretreatment — orange-red, green in polarized light
Nuclei
—
blue
Eosinophiles
—
bright red with Congo red
Note: reddish staining along collagen fibres is unspecific, in polarized light yellowish
color. By light microscopy, amyloid is an amorphous, homogeneous protein deposited
outside of cells!
To increase the sensitivity of Congo red stain use fluorescence microscope with
Rhodamine filter! (not as often falsly recommended: fluorescein filter!)
Von Kossa
Note: use thick sections 6-8µm only!
Staining procedure
1.
2.
3.
4.
5.
Dewax and bring sections to distilled water
Place sections in silver nitrate solution (5%) at daylight
Transfer section into sodium thiosulphate (5% )
Rinse in destilled water
Counterstain with nuclear fast red
60 min.
2 min.
5 min.
6. Wash in tap water
7. Dehydrate through alcohols, twice (96%), twice abs. Alcohol, xylol
8. Mount as usual (Pertex)
Solutions
Silver nitrate
5% aqueous, (Merck Art. 1.01512 (www.merck.de)
Sodium thiosulphate
5% aqueous (Merck Art. 1.06512) dissolved in 300 ml distilled water)
Mix before use
Nuclear fast red
Nuclear fast red (Chroma Art. 1A 402) 0.1 gr
Aluminium sulphate (5%), aqueous (Merck Art. 1.01102)
Boil, cool, filtrate
Results
Calcium salts
Nuclei
Cytoplasm
black
red
light pink
Note: von Kossa stains “phosphates” not calcium! To prove the calcium content use:
Alizarin Red S.
Alizarin Red S
Note: use thick sections 6-8µm only!
Staining procedure
1. Dewax and bring sections to distilled water
2. Place sections in alizarin red S solution for 30 seconds to 5 min.
3. Examine under the microscope every 30 sec. As soon as red color appears,
shake off excess stain. Do not counterstain!
4. Dehydrate and clear rapidly through acetone, acetone-xylol (1:1), xylol.
5. Mount as usual (Pertex)
Solutions
Alizarin Red S
Dissolve Alizarin red S 2 gr. in 100 ml distilled water
Adjust to pH 4.1-4.3 by ammonium hydroxide (25%) (drop by drop!). Stir constantly!
Results
Most calcium salts
Calcium oxalate
red
negative
Note: To avoid over staining, frequent control under the microscope is necessary!