Basic procedure of histopathology in lab

1. Histology: Lab Manual
Dr. Mandeep Kaur
PGGCG, Sector 42, Chandigarh
Mandeep.kaur15@hotmail.com

2. Requirements
Chemicals
• Ethanol
• Formalin
• Benzene
• Xylene
• Paraffin wax
• DPX
• H & E stain
• HCl & NH3
Glassware/instruments
• Coplin Jars
• Glass slides
• Beakers
• Wax dispenser
• Hot plate
• Water bath
• Rotary microtome
• L-shaped moulds
• Oven
• Microscope

3. Histology procedure
1. Fixation
2. Dehydration
3. Clearing
4. Infilteration
5. Embedding
6. Section cutting
7. Staining
8. Mounting

4. Histology Procedure
Fixation

5. Tissue Fixation
Tissues are fixed:
•To preserve cells and tissues constituents
•To prevent their degradation
Bouin’s fixative/formalin for 24 hrs
(with fixative’s volume 20 times greater)

6. Histology Procedure
Fixation Tissue Processing
a. Dehydration
b. Clearing
c. Infilteration

7. Dehydration
30%
50%
70%
70%
90%
Ab
Alcohal
3 min
3 min
3 min
overnight
1 hr in each grade
Remove fixative and water from the
tissues and replacing with dehydrating
fluid.

8. Clearing
Replacement with benzene
The tissues were shifted to varying concentrations of benzene and
alcohol in order to replace alcohol with benzene:
Absolute alcohol: Benzene:: 1:1 for 1 hr
Absolute alcohol: Benzene:: 1:3 for 1 hr
Pure benzene for 1 hr
Replacement of dehydrating agent with a fluid that is
totally miscible with both the dehydrating fluid and the
embedding media.

9. Benzene: wax: 1:1 for 30 min
Benzene: wax: 1:3 for 30 min
Immersed in molten paraffin wax
& two changes are given in 1 hr interval
Replacement with wax
Benzene from tissues was then replaced by wax as follows:
Infilteration
Replacement of clearing agent with embedding media.

10. Histology Procedure
Fixation Tissue Processing
a. Dehydration
b. Clearing
c. Infilteration
Tissue embedding

11. Embedding
Suitable sized tissue was embedded in molten wax
in L- shaped moulds and rectangular wax blocks are
prepared.
•Tissues are orientated in desired direction.
•provide sufficient external support during
sectioning

12. Histology Procedure
Fixation Tissue Processing
a. Dehydration
b. Impregnation
c. Clearing
Tissue embeddingsectioning

13. Sectioning
5 µ thin sections are obtained using rotary
microtome.
Stretching
Sections are fixed on glass slides with a very thin
coat of albumin and stretched on hot plate using
water.
Dewaxing
The sections on slides were dewaxed in
xylene and passed through descending series of alcohol
and finally in water (2-3 min in each).
30% 50% 70% 90%
Water

14. Histology Procedure
Fixation Tissue Processing
a. Dehydration
b. Impregnation
c. Clearing
Tissue embeddingsectioningstaining

15. Staining
Sections are stained with Haematoxylin for 15 min.
Differentiated in 1% HCl and NH3 solution for 2 min.
washed with water.
Sections were dehydrated in ascending series of alcohol
reaching upto 90% alcohol (2-3 min in each grade).
Sections were stained with Eosin for one min and
differentiated in 90% alcohol and dehydrated in absolute
alcohol (2-3 min).

16. Histology Procedure
Fixation
Tissue Processing
a. Dehydration
b. Impregnation
c. Clearing
Tissue
embedding
sectioningstaining
Mounting Viewing

17. Sections were passed through xylene for clearing wax and mounted in DPX

18. Histopathological studies of kidney
Fig: Photomicrographs (H & E) of Kidney of Ctenopharyngodon idellus
Abbreviations: DCT-Distal convulated tubule, PCT- Proximal convulated tubule, BC-Bowman’s capsule, CD-Collecting duct

19. Thank you