Staining techniques and troubleshooting Flashcards
General Staining
DEWAXATION - xylene is hydrophobic
▪Xylene min
▪ Xylene 2 min
REHYDRATION- descending grades of alcohol
▪ 100% alcohol2 min
▪ 95% alcohol 1 min
▪ 70% alcohol 1 min
▪ RTW or dH2O 2 min
STAINING HAPPENS HERE
DEHYDRATION
▪ 95% alcohol 5-15dips
▪ 100% alcohol 5-15dips
▪ 100% alcohol 5-15dip
CLEARING
▪Xylene 1 min
▪ Xylene 1 min
Reagent Set what do they contain
how to handle
- contain volatile and toxic vapors keep in FLAMMABLE cabinet
-ensure ventilation is turned on
-have proper PPE
-Label slides prior to staining and put a permenant label on after staining
-put slides in a carrier when transferring
-when using the slide carrier make sure its dry.If you are going to take the slides into the water
bucket, it must be xylene-free. If you are taking
the slides to xylene, it must be water-free. Water
and xylene are not miscible. It will contaminate
the reagents. If the slide carrier is wet, dip it into
a fresh bucket of 100% alcohol. It is miscible with
both water and xylene.
What is the purpose of staining? and what are the THREE MAJOR STEPS IN STAINING PROCESS
To aid in the visualizing tissue components.
To demonstrate tissue components differently
- DEWAXATION AND REHYDRATION: “Taking sections to water”
- STAINING: Applying reagents and stains/dye.
- DCM: Dehydrate, Clear and Mount.
What is DEWAXATION AND REHYDRATION
-dewax in xylene as it removes infiltration medium (paraffin) so the tissue is prepped for staining
-the paraffin wax remaining after being taken out of the oven will be removed by xylene
-descending grades of alcohols from 100-95-70 clear out the xylene and introduce the tissue to water with RTW in a pre filled bucket
-use a low flow rate so tissue doesnt break off and all alcohol is rinsed off the slide
What happens in the staining phase
-apply reagents in aqueous environment
-after each step in staining reagent the slides have to be rinsed out in RTW bucket
-After blueing with Scott’s TWS and RTW, take your
slide to the microscope and check to make sure
the nuclei are blue and not red.
What are you check for when you CHECK DIFFERENTIATION MICROSCOPICALLY
-in the bluing step the red color of HX is converted into a blue color
-alkaline pH of bluing solution causes a mordant dye-lake to reform in the tissue and become more permanent.
-dont let slides dry out - take it out of the water and put it on the mic
-if slide if left out for too long itll look grainy. dip it back into water and rehydrate to check microscopically
What is the DEHYDRATION phase
slides are preserved by removing water/aqueous stains by process of DCM
Ascending grades of alcohol.
Gradual removal of water from tissue.
Very important to avoid contamination of alcohols and xylenes
What is the CLEARING phase
Removal of alcohol from tissue slides.
Has high refractive index .
Tissues are left clear and crisp without a cloudy haze over tissue (unless contaminated.
Xylene must not be contaminated with water
(immiscible with each other).W
What is the purpose of mounting
-protect stained tissue from damage
-better for microscopic examination stops refraction of light under the mic
-preserves for storage
-need glass coverslips
-permount mounting media - make sure to check viscosity. If exposed to air itll harden ; add drops of xylene to dilute if too thin add permount reagent
-wear ppe and work in ventilated area
-make sure there is no haze
-opaque spots show incomplete dehy and clearing
-wipe frosted side first to check where the tissue is
-dont allow tissue section to dry out, return slides to xylene if dried out
-lower coverslip at 45 degree angle, permount is drawn up by capillary action and dab to remove excess mountant on paper towel
staining results what you should see - microscopic examination
Nuclei Blue
Muscle and collage - pink
RBCs Orange-pink
do tissue id
REAGENTS IN THE DEWAXATION SET
what are their properties
- XYLENE - HYDROPHOBIC
- ALCOHOL - HYDROPHILIC
- WATER
- Which bucket do the slides go into first?
- What would happen if the slides were placed in
the wrong reagent…what is your next course of
action?
xylene
take slide and dip into 100% alcohol work back 70, 90, 100 and back into xylene to dewax
not anyother grade because itll have water in it
-dry slide carrier or only one that has been 100% alcohol
What would you do if slides were exposed to
water first?
dip in 100% alcohol
What would you do if slides were exposed to
rehydrating alcohol first? 100%, 95%, 70%, and not
xylene
dip in 100% alcohol
What would you do if slides were not dewaxed or
rehydrated and stain was applied to the tissue?
rinse in water, remove the H&E stain, then in ascending grades of alcohol, xylene to dewax and follow steps as normal
tissue is not affected because there is still wax on the tissue