Histotechnology
Technical methodsLaboratory Method
STAINING RESIN SECTIONS OF BONE
For routine examination of undecalcified bone for metabolic bone slides, the following techniques are employed. Sections are stained free-floating without etching the resin.
i) Von Kossa/Van Gieson
ii) Goldner trichrome (personal modification)
iii) Toluidine blue
iv) Perls' Prussian blue
VON KOSSA/VAN GIESON
1. Sections to distilled water x 2 for 15 minutes.
2. Place sections into a clean small glass beaker of 1% silver nitrate (filter solution) and expose to UV light for 30 minutes.
3. Wash in distilled water x 2 for 10 minutes.
4. Fix in 1% solution thiosulphate for 30 seconds
5. Repeat step 3.
6. Counterstain in Van Gieson (Unna's variant) saturated with picric acid for 15 minutes.
7. Rinse in distilled water.
8. Blot dry.
9. Rinse in alcohol x 2.
10. Wash in methyl cyclohexane x 2.
11. Mount in Picromount.
RESULTS
--Mineralised bone Black --Osteoid/collagen Red --Marrow/muscle Yellow
Solutions
VAN GIESON (UNNA VARIANT)
--Acid fuchsin 2.5g --Nitric acid (conc) 5ml --Glycerine 100ml --Distilled water 900ml --Picric acid to saturation
GOLDNER TRICHROME (MODIFICATION)
1. Sections to distilled water x 2 15 minutes
2. Place sections in Weigert's haematoxylin 20 minutes
3. Wash in water.
4. Differentiate with 0.5% acid alcohol.
5. Wash in water 20 minutes
6. Ponceau/acid fuchsin/azophloxine 5 minutes
7. Rinse in 1% acetic acid 10 seconds
8. Phosphomolybdic acid/orange G 20 minutes
9. Repeat step 7
10. Light green 5 minutes
11. Rinse in water
12. Blot dry
13. Rinse in alcohol x 2
14. Wash in methyl cyclohexane x 2
15. Mount in Picomount
RESULTS
Nuclei Blue/black Mineralised bone/muscle Green Osteoid/collagen Red
Solutions
WEIGERT'S HAEMATOXYLIN
SOLUTION A
--Haematoxylin 10g
--Distilled water 1000ml
Ripen for at least 2 weeks before use
SOLUTION B
--Ferric chloride (hydrated) 11.6g
--Distilled water 1000ml
--2% hydrochloric acid 10ml
For use mix equal points of A and B immediately before required. Do not keep working solution pre-made.
PONCEAU DE XYLIDINE/ACID FUCHSIN
--Ponceau de xylidine 1.5g
--Acid fuchsin 0.5g
--Acetic acid (conc) 2ml
--Distilled 98ml
AZOPHLOXINE
--Azophloxine 0.5g
--Acetic acid (conc) 0.6ml
--Distilled water 99.4ml
PONCEAU/ACID FUCHSIN/AZOPHLOXINE (WORKING SOLUTION)
--Ponceau de Xylidine/acid fuchsin 12ml
--Azophloxine 8ml
--0.2% Acetic acid 80ml
Re-use and keep made up.
PHOSPHOMOLYBDIC ACID/ORANGE G
--Phosphomolybolic acid 6g
--Orange G 4g
--Distilled water 1000ml
LIGHT GREEN
--Light green 2g
--Acetic acid (conc) 2ml
--Distilled water 1000ml
TOLUIDINE BLUE
1. Sections to distilled water x 2 15 minutes
2. Toluidine blue/EDTA 1 hour
3. Wash in distilled water
4. Blot dry (37°C overnight or 60°C 8 hours)
5. Rinse in methyl cyclohexane
6. Mount in Picomount
RESULTS
--Nuclei Blue
--Mineralised bone Light purple
--Osteoid Colourless - pale blue
--Mineralised front Light blue
--Reversal line Dark blue/purple
Solutions
TOLUIDINE BLUE
--Toluidine blue 1g
--Diaminoethanetetra - acetic acid disodium salt 5g
--Distilled water 100ml
PERLS PRUSSIAN BLUE
1. Sections to distilled water x 2 15 minutes
2. Perls reagent freshly prepared 40 minutes
3. Wash in distilled water
4. Counterstain in 1% neutral red 30 seconds
5. Washing in distilled water
6. Blot dry
7. Rinse in alcohol x 2
8. Wash in methyl cyclohexane
9. Mount in Picomount
RESULTS
--Ferric iron Blue
--Nuclei Red
PERLS REAGENT
--Potassium ferrocyanide 1g
--1% hydrochloric acid 50ml
SECTIONS LABELLED WITH TETRACYCLINE
1. Carefully remove sections which have been stored in 70% alcohol and transfer to 100% alcohol.
2. Wash in fresh 100% alcohol.
3. Wash in methyl cyclohexane x 2.
4. Mount.
FLATTENING FREE-FLOATING SECTIONS 1. Carefully pick up the sections individually with fine forceps and place in water in a clean container.
2. Place the section carefully on the wall of the container by floating the section into position.
3. Carefully blot dry.
4. Carefully tease the section off the wall and proceed as previously described.
MOUNTING FREE-FLOATING SECTIONS
1. Carefully pick up the sections from methyl cyclohexane and cut off excess resin with a pair of small scissors.
2. Place section on an aliquot of Picomount which has been dispensed in the centre of a clean slide with the aid of a glass syringe.
3. Dispense further mounting medium to cover the section.
4. Cover the section with an appropriate size cover glass ensuring that any air bubbles are removed.
5. Carefully wrap the slide in a polythene envelope provided.
6. Place 1 or 2 packing slides on top of the envelope.
7. Carefully clamp with a bull-dog clip.
8. Dry overnight in a 37°C oven.
CLEANING SLIDES
1. Remove slides from 37°C oven and allow them to cool to room temperature.
2. Remove carefully, clip, packing slides and polythene envelope.
3. Place slides in xylene to clean off excess mountant. Use a fume cupboard.
4. Wipe slides clean and dry.
NOTE: No responsibility is assumed by The University of Nottingham or the Queens Medical Centre NHS Trust for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. It is the users responsibilty to ensure that all procedures are carried out according to appropriate Health and Safety requirements.
© Copyright 1997 University of Nottingham Medical School Division of Histopathology. This page was last built on Mon, May 5, 1997 with Frontier. Thanks for looking in. Comments to James.Lowe@nottingham.ac.uk