PRACTICAL EXERCISE
This lab report well be prepared should comprise of 6 pieces of A4, size 12 font or larger includes 3 sections:
A) Introduction:
1) Talk about the renal disease and the role of cellular pathology for investigating it.
2) Talk about Amyloid and the role of cellular pathology for investigating it. Provide table demonstrate types of amyloid
3) Provide principle and the important of these stain in investigation amyloid and state some example diseases that each stain used to investigate :
H and E :
PAS stain:
Cango red:
Sirius red :
4) Aim of this report is to examine the main ways of investigation renal disease using the following stains ( H&E , PAS, Cango red and Sirius red . and how each one contribute different to each diagnosis.
B) Material and Methods :
HAEMATOXYLIN & EOSIN (H & E)
1. Sections to water,
2. Stain in Mayer’s haemalum 1 minute
3. Blue in tap water 5 minutes
4. Stain in Eosin 2 minutes
5. Rinse in tap water, dehydrate in industrial methylated spirit (IMS), clear in Histoclear and mount in DPX.
PERIODIC ACID – SCHIFF (PAS)
1) Section to water
2) Rinse in distilled water
3) Oxidise in 0.5% periodic acid – 10 minutes
4) Rinse in distilled water then thoroughly (at least five minutes) in running tap water
5) Rinse in distilled water
6) Treat in Schiff reagent – 20 minutes
7) Rinse well in tap water for at least 5 minutes
8) Stain in Mayer’s haemalum – 1 minute
9) Blue in tap water – 5 to 10 minutes
10) Dehydrate in ethanol, clear and mount in DPX
Notes
All solutions are made up in distilled water. The use of any counterstain other than a weak haematoxylin may mask results. Schiff reagent may be prepared in a variety of ways and a reference text should be consulted.
CONGO RED (HIGHMAN’S MODIFICATION)
1) Section to water
2) Stain in Highman’s congo red1 – 5 minutes
3) Rinse and differentiate in Highman’s differentiator2 – 1 minute
4) Rinse in tap water
5) Stain in Mayer’s haemalum – 1 minute
6) Blue in tap water
7) Dehydrate, clear and mount in DPX
Notes
1. 0.5% congo red in 50% alcohol
2. 0.2% potassium hydroxide in 80% alcohol
SIRIUS RED FOR AMYLOID
1) Section to water
2) Stain in Mayer’s haemalum – 1 minute
3) Rinse and blue in tap water – 10 minutes
4) Rinse in 70% alcohol
5) Stain in sirius red solution1 in a Coplin jar – 1 hour
6) Wash well in tap water
7) Dehydrate, clear and mount in DPX
Notes
Dissolve 0.5g sirius red F3B in 45 ml of distilled water. Add 50 ml of absolute ethanol, mix, and add 1.0 ml of 1% sodium hydroxide. Swirl vigorously whilst adding 4.0 ml of 20% sodium chloride. Allow to stand overnight then filter.
C) Results :
i)Examine the stained slides and describe the results obtained with each method, with particular focus on the staining of the renal glomerulii. Explain the staining results achieved for each method in the context of biological stain theory. Describe how these methods are utilised in the diagnostic histopathology laboratory in the investigation of renal disease, detailing how each stain might aid in the diagnosis. Besides these stains and those for amyloid, name 2 other histological staining methods that might be useful in these investigations, why they might be useful, and the theory behind how they differentially stain the components of interest. Name two other types of test routinely carried out in many histopathology departments which might help in the diagnosis of renal disease but which don’t involve histological stains(dyes), describe what they demonstrate and the relevance to the diagnosis.
ii) Demonstrate amyloid in the wax sections, describe the results seen and the relevance of amyloid to the investigation of renal disease. How could the validity of the test be improved?
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These are some answers of these questions :
H & E:
– Most widely used stain in medical diagnosis
– Application of the basic dye hematoxylin, which
. colors basophilic structures with blue-purple hue, and
. alcoholic- based acidic eosin Y, which colors eosinophilic structures bright pink.
– The basophilic structures – intracellular or extracellular protein. The lewy bodies and Mallory bodies are examples eosinophilic structures. Most of the cytoplasm is eosinophiic. Red blood cells are stained intensely red.
– Some structures do not stain well :
. BM: PAS stain or Silver stain
. reticular fibers : silver stain
. hydrophobic structures also tend to remain clear ; these are usually rich in fat, eg. Adipocytes, myelin, and golgi apparatus membranes.
PAS stained Kidney slide:
This normal glomerulus is stained with PAS to highlighted basement membranes of glomeruls capillary loops and tubular epithilum. The capillary loops of this normal glomerulus are well-defined and thin. The endothelial cells are seen in capillary loops. The mesangial regions are of normal size. Podocytes are present and forming the visceral epithelial surface. Bowman’s space is seen along with parietal epithelial cells.
PAS:
– To identify glycogen in tissues.
– Reaction of periodic acid selectively oxidizes the glucose residues, creates aldehydes that react with the Schiff reagent and creats a purple-magenta color.
– A suitable basic stain is often used as a counterstain.
– Mainly used for staining structures containing a high proportion of carbohydrate marcomoleculecules ( glycogen, glycoprotein, proteoglycans), typically found in eg. Connective tissues, mucus, and basal laminae.
name 2 other histological staining methods that might be useful in these investigations:
1) Masson’s trichrome
– Three color staining protocol.
. red keratin and muscle fibers
. blue or green collagen and bone.
. light red or pink cytoplasm and
. dark brown to black cell nuclei
– The trichrome is applied by immersion of the fixated sample into weigert’s iron hematoxylin , and then three different solutions, labelled A,B and C.
• Weigert’s hematoxylin is a sequence of three solutions: ferric chloride in diluted hydrochloric acid, hematoxylin in 95% ethanol, and potassium ferricyanide solution alkalized by solution borate. It used to stain the nuclei .
2) Silver stain
Share with PAS to investigate some disease
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