Microtechnique

Lecture 2
Dr. Esam Qnais
Hashemite University
 Washing
Before dehydration we have to wash the -
.fixative
Aim of washing: to remove excess fixative -
.which might interferes with subsequent steps
Tap water, buffer and 70% alcohol are used for
.washing
 Dehydration
Dehydration: gradual removal of water from the
tissue using ascending grads of ethyl alcohol
.to prevent tissue shrinking
100% ,95% ,90% ,70% ,50% ,30%
C1V1=C2V2
Good dehydration no white ppt during
clearing
 Dehydrants
(Dehydrating agents)
Ethanol: rapid but might cause shrinkage- 1
2- Methanol: very expensive and causes less
shrinkage
3- Isopropanol: slow and causes less shrinkage
4- Acetone: rapid, highly flammable, expensive
.but causes less shrinkage
: Clearing
Replacement of alcohol in tissue by clearing - -
fluid like xylene, benzene, or acetone
An intermediate step between dehydration and -
.infiltration
Achieved by using a reagent that is miscible in -
.both dehydrant and infiltration reagent
They make tissue transparent (clear) -
They increase the index of refraction -
:Clearing media (agents)
Xylene: good one, most common, but it causes hardening of- 1
.the tissue if it uses for long time. Avoid inhalation of vapour
Benzene: volatile, flammable and it leaves air pockets- 2
Cedarwood oil: expensive, works at slower rate, partly- 3
.miscible with methanol
Chloroform: often used mixed with benzene- 4
:Criteria of good clearing media
().Price- 1
().Volatility- 2
().Rate of clearing- 3
().Rate of removable during infiltration- 4
Dissecting tools
Rotary microtomes
Paraffin ovens
Embedding moulds
Paraffin block holders
Sections floating baths
Microscope slides
Cover slips
Coplin jars
Staining boxes and staining assembly
Slides boxes
Camel hair brush
Wax dispenser
Glassware (vial, dropping bottle, etc)
 Dissecting tools

Camel hair brush

Vial

Dropping bottle

Microscope slides
 Paraffin ovens

Rotary microtomes

Paraffin block holders

Embedding moulds
Coplin jars
Paraffin block holders
 Wax dispenser

Hot plate
Sections floating baths
Slides boxes
staining and in situ hybridization C: Coplin jar; D: staining
dish with slots. Adapted from Preece (1972).