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
WATER AND SLIDES TURN MILKY WHEN
SLIDES ARE PLACED IN WATER FOLLOWING ALCOHOL DURING DEPARAFFINIZATION
What would cause this and the Corrective action
-Presence of xylene on the slides.
-Inadequate time in rehydrating alcohols to
remove xylene.
CORRECTIVE ACTION:
* Change the alcohols in the rehydration section of the dewaxing set.
* Then take the slides through fresh 100% alcohol → 95% alcohol → 70% alcohol → water
how will CONTAMINATED REAGENTS IN THE
DEWAXATON SET affect staining
- Can cause poor quality staining of tissue
components. - Can obscure the microscopic image of the tissue.
*can be caused by inadequate rinsing
in xylene you can see contaminated water droplets
LIDES ARE HAZY OR MILKY IN THE LAST XYLENE BEFORE COVERSLIPPING causes and corrective action
Causes:
-Incomplete dehydration in alcohols during DCM.
-Contaminated 100% alcohol that carried water
over to the xylene buckets in DCM.
CORRECTIVE ACTION:
-Back up the slides and change the fresh alcohols and xylene solutions.
* Then re-dehydrate in the alcohols and clear the sections in xylene.
* The slides should be clear and transparent.
LOW REAGENT LEVEL can lead to
-Inadequate dewaxation. If paraffin is not removed from tissue stains will not be able to penetrate
-incomplete rehydration and dehydration in DCM because of the contaminated 100%etoh and xylene
-make sure reagents are filled to the reagent line on bucket and you may need to repeat dewaxation and rehydration steps
how to troubleshoot staining problems
-check staining of slides when they are in the last xylene bucket before coverslipping.
-skip coverslip removal step if troubleshooting is needed
-if the slides can be troubleshooted “take them back to water”
if you need to take back to water - work backwards from DCM
xylene → xylene → 100% ETOH → 100% ETOH → 95% ETOH → RTW → troubleshoot stain
ARTIFACTS ON MOUNTED SECTIONS - not due to staining
- WATER DROPLETS
- WHITE SPOTS IN TISSUE SECTION – PATCHY STAINING
- DRY MOUNTING: CORN-FLAKING OR TRAPPED AIR
- RETRACTION
- TISSUE NOT CRISP
- BUBBLES FROM COVERSLIPPING
- MOUNTANT ON THE COVERSLIP
- SCRATCHES ON THE TISSUE/TISSUE WIPED OFF.
What causes water droplets and how to correct
Cause
* Incomplete dehydration.
* Contaminated 100% alcohol in DCM.
* Contaminated xylene
*can look like color leaching
CORRECTIVE ACTION:
* remove coverslip and Soak slide in xylene to remove coverslip.
*xylene will dissolve permount
* Change contaminated reagents – take slides to fresh 100% ETOH and clear in fresh xylene 2x – dip slides until they are no longer streaky.
* Ensure there are no water droplets in xylene.
WHITE SPOTS IN TISSUE SECTION
causes and corrective action
Causes
* Incomplete deparaffinization and dewaxation.
* Patchy staining due to presence of wax in the tissue prior to application of stains – irregular staining.
* Contaminated dewaxing xylene.
* Section not dried properly in the oven.
CORRECTIVE ACTION:
* Ensure slides stay longer in the dewaxing xylene.
* Avoid using dewaxing xylene contaminated with water.
* Remove stain, remove wax and re-stain.
* make sure slide is completely dry in the oven
CORN FLAKING OR TRAPPED AIR - dry mounting artifact
causes and corrective action
causes
Too slow in applying the coverslip.
-trapped air in nuclei = cornflake artifact
-high refractile lines in tissue because its dried out BEFORE coverslipping
- ialso Xylene has evaporated from tissue prior to
coverslipping.
CORRECTIVE ACTION:
* Soak slide in xylene until the coverslip comes off.
* Re-coverslip making sure xylene is not dried out
RETRACTION
causes and corrective action
causes
-Mounting medium was too thin (too much xylene was added to thin it out) can also cause bubbles under the coverslip
corrective action
-Soak slide in xylene until the coverslip comes off.
-Re-coverslip with a mounting medium that has the correct consistency.
TISSUE SECTION NOT AS CRISP AS USUAL
causes and ca
CAUSES:
* Mounting medium too thick.
CORRECTIVE ACTION:
* Re-coverslip with correct coverslipping technique and correct mountant medium consistency
BUBBLES FROM COVERSLIPPING
causes and ca
causes
* Incorrect angle when bringing slide close to
coverslip.
* Coverslipping too fast
CORRECTIVE ACTION:
* Soak slide in xylene until the coverslip comes off.
* Re-coverslip and lower slide gently over the coverslip on a 45° angle.
-the tissue can be seen as dark and grainy
EXCESSIVE MOUNTING MEDIA causes and corrective action
-foggy
causes
* Too much mounting medium applied. one drop is good
-make sure its not too thick with clean and dry gloves
* Incorrect handling of coverslip - only the edges
* Mountant medium on gloves.
corrective action
* Soak slide in xylene until the coverslip comes off.
* Re-coverslip making sure you dont smudge the new coverslip.
SCRATCHES ON TISSUE/WIPE OFF
causes and corrective action
causes
-improper handling of tissue during staining
-tissue was too close and touched other materials causing it to be wiped off during coverslip or staining. if it got close to staining racks
CA
-handle tissues with care
-put tissue in center of slide when mounting onto slide during microtomy
-cant troubleshoot -tissue needs to be recut and restained
