Lecture 11 Flashcards
What is the purpose of staining
-show the tissue components
-show the difference in tissue components
What are the six major steps in staining
Take sections to water
-dewaxation - removal of wax with xylene both are hydrophobic
-rehydration - gradual introduction of water to clear out xylene (descending OH, then RTW or dh2o
Apply reagents and stains
-staining - application of staining solutions
-dehydration-gradual removal of water- Ascending grades of alcohol
Dehydrate, Clear, Mount
-dehydration - slides passed through ascending order of alcohol 95%, 100%, 100% xylol, xylol. Adequate dips must be done to make sure water is removed by the time the slides reach the 2nd alcohol (5-15)
-clearing -removal of alcohol, clearing the tissue making it transparent with xylene
-mounting - using resinous or aqueous mountant . To protects the tissue and stop refraction of light when viewed under the microscope
-slides should never dry out during any of the staining steps
What is the purpose of coverslipping and what is needed
-protect and preserve the section that has been affixed to a microscope slide
- to give better optical quality when looking at samples under the microscope
-need mounting medium and coverslip
-resinous mounting medium stored in glass pots and dropper bottles , thin out with xylene when it becomes viscous
-if too thin we dont use it and make up a new permount
-dont introduce bubbles or they will appear at artifact on sample
What is the ideal mount medium
-solution that covers the tissue section, and should harden into a permanent mount
- Permount (water insoluble) – paraffin sections - paramount
- Aqueous (water soluble) – frozen sections - glycerol - fatty tissues
What are the properties of a good mounting medium
- Refractive index is close to coverglass (1.518)
- Non-reactive with the tissue section.
- Stable over time without crystalizing, darkening or changing its refractive index.
- Stable enough so it doesnt dissolve in xylene or water (the solvent used for permount and glycerol).
- Does not cause the stain on the tissue to fade or leach.
- It must solidify/dry/harden in a short time.
What are the types of mounting media
CANADA BALSAM – Natural resin
* dissolves in xylene
* yellows on storage
* Dries slow
* Causes dyes to fade
PERMOUNT – Synthetic resin
* Insoluble in xylene
* Dries rapidly
* Neutral ph – will not cause dyes to fade over time.
What are aqueous mountants and their uses
- Glycerin
- Glycerin jelly
- Farrant’s medium
- Sugar mixture
- RI =1.46-1.47
- Uses:
- Frozen section
- Staining for lipids using frozen sections
- Histochemical reactions
- Metachromasia
how to store aqueous mounted sections
-Use nail polish or paraffin wax to ring the mountant beneath the coverslip.
* To prevent evaporation
What is a coverslip and why is it used
*thin piece of high quality pre-cleaned glass
* made of silicate
* many sizes and thicknesses
* Either square or rectangular
Are placed over the section to:
* Keep the section flat
* Hold the section in place
* Protect the section from dust and accidental damage
* Retard dehydration and oxidation of the section
* Protect the microscope’s objective lens
thickness - 0.13 – 0.16 MM
-if too thick then the image will not be clear
precautions for coverslipping
-under a well-ventilated fume hood to avoid inhaling toxic vapors. - like those from xylene
-safety glasses should be worn during coverslipping
* Wear gloves or forceps may be used to remove slides from xylene to avoid irritation and drying of skin.
What is CELLOIDINIZATION
-protective coat (prevents tissue from falling off)
-Used in alkaline silver staining techniques (after rehydration step).
- To protect consult slides during transport.
-Promotes tissue attachment
FORMALIN PIGMENT
what does it look like
formation
removal
-brown , doubly refractile deposit seen both intra/extracellularly in tissues fixed with simple formalin - formal saline aka acid formaldehyde hematin
formation- Stored un-buffered, formalin forms formic acid that reacts with the heme of the hemoglobin molecule.
-can form quickly if tissue is bloody
removal of formalin pigment: Rinse with picric acid after 100% alcohol in “hydration” step. leaves yellow color to tissue.
how to remove the yellow discoloration before the tissues get stained
- Wash in RTW for 20-30 min
- Acid alcohol + wash in RTW
- Prolonged alcohol wash
- Lithium carbonate in 70% alcohol
Mercury pigment
appearance and formation
- APPEARANCE: dark brown to black crystals in the tissue.
- FORMATION: precipitates as mercury salt on the tissue section.
how to remove mercury during dehydration (during tissue processing)
- Add 20 ml of strong alcoholic iodine to each litre of the first 70% or higher ethanol (final concentration is 0.4%).
- The brown discolouration will be removed in later ethanol steps.
- Continue with processing
