ADHESIVE, MOUNTING & LABELLING

• Adhesives

• Mounting

• Labeling

● (______): to keep sections attached

● (_______): to preserve and prepare slides

● (_________): to ensure proper identification

(FISHING OUT) (10°C lower than a melting point) ( 10 degrees Celsius lower) (distort or disintegrate) (water bath) (fished out and drained) (water) (float freely) (excess water)

● Done after sections are floated out on a water bath

○ water bath should have a temperature that is (______________________) of the wax

■ It is important that the water bath temperature is maintained at

about (_______________) than the melting point of the wax.

■ This temperature control ensures that the tissue sections do not (_____________) during transfer.

○ Slide is immersed in the (________) and the section is (____________)

■ Once the bath is set, immerse the slide into the (______), allowing the section to (____________).

■ Then gently fish out the section, ensuring that it is properly drained of (___________).

(ORIENTATION) (section/ribbon) (verify the correct orientation) (Proper positioning) (diagnostic process) () ()

● Correct positioning of the tissue (_________) on the slide

○ Following fishing out, the next critical step is to (______________) of the tissue section or ribbon on the slide.

● (____________) is essential for consistent staining and accurate microscopic evaluation

● Any misalignment at this stage could complicate the (______________).

(DEPARAFFINIZATION & DRYING OUT) ( residual wax) (firmly attached)

● In this section, we cover the methods used to depyrophinize and dry out tissue sections before staining.

● The main goal is to remove any (_________) and ensure that the tissue is (__________) to the slide, which is crucial for consistent and accurate staining.

(Wax oven:) (Incubators) (Hot plate) (Alcohol lamp / bunsen flame:) (Delicate tissues) (onger drying time at lower temperature) (Blower-type electric slide dryer:) (Blower-type electric slide dryer)

● (_________) 56°C - 60°C for 2 hours

● (_________): 37°C (overnight)

● (_________): 45°C - 55°C for 30-45 mins.

● (_____________) used for rapid drying, but caution must be taken to avoid overheating or damaging the tissue

● (_________): 37°C for at least 24 hours

○ For more delicate tissues like your CNS tissue or brain, a (___________________) set at 37 degrees Celsius for 24 hours or longer is recommended to avoid splitting and cracking of the section due to excess heat

(_______________) 50-55°C for 20-30 mins

○ uses controlled airflow and temperature to efficiently dry the slides avoid splitting and cracking of the section due to excess heat

● (__________________________): 50-55°C for 20-30 mins

○ uses controlled airflow and temperature to efficiently dry the slides

(ADHESIVES) (prevent washing) (routine staining) clean, grease-free slides) (urgent cryostat sections) (central nervous system) (blood clot) (decalcified) (high temperatures) (integrity of the tissue ) () () () ()

● Alternative to drying

● Performed to prevent (_______) of tissue sections during staining, ensuring that even under rigorous staining conditions, the tissue remains firmly attached to the slide.

● It is a substance which can be smeared onto the slides so that the sections stick to the slides

● Not necessary for (_____________)

● Essential for methods that require exposure of sections to acids and alkalis during staining

● In these situations, adhesives help maintain the integrity of the tissue section throughout the staining process

● While routine staining might not require additional adhesives, their use is critical in methods where tissue sections are subjected to conditions that could otherwise cause them to detach

● If (___________) are used and sections are adequately dried, the sections will not float off during staining, and adhesive will not be necessary.

● Instances when sections may float from slide:

○ For (_________) to be submitted for immunocytochemistry

○ For (____________) tissues

○ For tissues containing (______)

○ For tissues which have been (_______)

○ When sections are to be subjected to (_____________)

○ For these scenarios, the use of adhesives is recommended to ensure the (_______________) sections during the staining process

(MAYER’S EGG ALBUMIN) (Egg white 50 ml) (Glycerin 50 ml) (Thymol) (make, convenient, and inexpensive. ) ( dirty background) () () () () () () () () () () () () () () () ()

(________________)

● Most commonly used

● Components:

○ (_____________)

○ (___________)

○ (_________) - to prevent the growth of molds, usually smeared into the clean glass slide before sections are oriented

● Advantage - very easy to (____________________)

● Disadvantage - it retains some of the stain and gives a (__________).

DRIED ALBUMIN

● Dried Albumin + NaCl + Thymol

(______________)

(_________________________)

● Sections are dried and stored in 70% alcohol until it is ready for staining

1% GELATIN

● Gelatin + Glycerol + Phenol

(__________)

(______________________)

● Added to the water bath during flotation rather than

applying it on slides

GELATIN-FORMALDEHYDE MIXTURE

● Gelatin + Formaldehyde

(_____________________________)

(___________________)

● Slides are coated and allowed to dry at 37°C for one hour or overnight prior to use

(POLY-L-LYSINE) (immunochemistry)

(__________________)

● Aqueous detergent diluted to 0.01% concentration widely used in (_______________)

● It is effective due to its positive charge, which promotes adhesion

APES (3-AMINOPROPYLTRIETHOXYSILANE)

● Very useful in cytology, particularly for cytospin preparations of proteinaceous or bloody material

● Best section adhesive available, and coated slides can be stored for a long time.

(MOUNTING MEDIUM) (physical damage, oxidation, and fading) (light refraction) (length dispersion) (transparent medium) (Binds specimen) (permanent use) (distortion of image)

(_________________)

● Mounting involves applying a (1) (__________) and placing a cover slip over the stained section

● This process is very essential because it preserves the staining by protecting the tissue from (_________________) over time

● If an unmounted stained section is observed under the microscope, differences in (__________) between the glass slide and tissue components may cause variations in (___________), resulting in limited microscopic detail.

● Hence, should be impregnated with (___________) with index of refraction close to that of glass and tissue

● (___________), slide and coverslip

● Preserve stained sections for (___________)

● Helps prevent the (______________) during examination.

REFRACTIVE INDEX (R.I.)

● enhance specimen details or make them invisible

● It governs the contrast between cellular detail and background and transparency of sample against bright field of microscope.

(FUNCTIONS OF MOUNTING) (RI higher) (Protects) (maximum degree of transparency) (Reduce) (permanent) (Ease)

(______________)

● (_________) than mounted sample for transparency

● Process that involves the use of a medium and a coverslip to facilitate the following:

○ (______) the specimen from damage

○ Provide (___________________) to stained tissue sections

○ (_________) the bleaching or deterioration from oxidation

○ For (_________) keeping

○ (______) of handling and storage of the slides

(center) (placed on the edge) (inclined ) (Gently pressed) (37℃ for 12-24 hours) (immersion oil) (store mounted slides vertically) (2 days) (Excessive mounting medium) (Excessive blotting) (For remounting) (Insufficient mounting medium) (forceps or a mounting needle) (50℃ for 2

hours.)

MOUNTING PROCESS

1. A drop of mounting medium is placed down the (_______) of the slide.

2. A clean, dry cover glass is (___________) of the slide and gradually (______) downward until it touches the mounting medium

3. (__________) on to the slide while the mounting medium quickly spreads through the whole area of the section.

4. Slide will be incubated at (_____________) after mounting to harden the medium

PRECAUTIONS:

● DO NOT USE (_______) on an uncovered slide.

● If in a hurry to view specimen, mount the slip and

handle the mount carefully and horizontally.

● DO NOT (_________________) for (______) if

cured at room temperature.

● (___________________) may ooze out from the

edges of the cover glass and should be carefully

wiped off using a fine cloth moistened with xylene.

● (___________), will dry up the section, causing

shrinkage and cracking.

● (_________), cover slip may be removed by

soaking in xylene.

● (_______________) may cause coverslip

misalignment or bubble formation.

● Bubbles can be removed by gently pressing with

(_________________).

● Setting can be accelerated by heating at (_______________)

(1.518) (Colorless and Transparent ) (xylene and toluene ) (consistency and harden relatively) (physical damage and chemical activity) (contamination ) (shrinkage and distortion of tissues ) (stain or affect staining) (color or pH) (adhesives) (crystallizing, cracking or shrinking) (stable permanent section ) () () () ()

CHARACTERISTICS OF A GOOD MOUNTING MEDIUM

1. Refractive index is close to the RI of glass (________)

2. (_________________)

3. Freely miscible with (__________)

4. It should not dry to a non-stick (_______________________) quickly

5. Protect from (____________________) (oxidation and changes in pH)

6. Resistant to (____________)

7. It should not cause (_____________________)

8. It should not leach out any (__________________)

9. It should not change in (___________)

10. It should be compatible with the (________) in use

11. It should set without (__________________)

12. Once set, it must produce a (__________________)

(RINGING) (fluid or semi-fluid mounts) (evaporation) (Immobilize) (sticking together) (KRONIG CEMENT) (DUROFIX)

(_________________)

● Process of of sealing the margins of the cover slip on

a mounted slide

● Functions:

1. Prevent the escape of (______________)

2. Prevent (_________) of mountant

3. (__________) the coverslip

4. Prevent the (____________) of the slides upon storage

● Materials:

○ (__________________): 2 parts paraffin + 4-9 parts powdered colophonium resin

○ (_______________): cellulose adhesive

(Water-based) (Organic solvent-based) (Lower 1.34-1.41) (Higher 1.51) (does not require) (requires) (Temporary) (Permanent) (shrinkage and

distortion) (water-soluble) (quick application) (long-term preservation) (Higher refractive index) (archival storage) (dry out overtime) (optimal clarity) (dehydration and clearing) (xylene exposure) (Hydrophilic stains) (Routine histology) (Synthetic resins) (undecalcified bones) () () () () () ()

TYPES OF MOUNTING MEDIA

	AQUEOUS MOUNTING MEDIA	NON-AQUEOUS/RESINOUS MOUNTING MEDIA
COMPOSITION	(__________) (glycerol, polyvinyl alcohol, sugar polymers)	(____________) (xylene-based resins like DPX, Canada Balsam)
REFRACTIVE INDEX	(_________), closer to water	(_________), closer to glass

SOLUBILITY	Water-soluble, (_______) dehydration or clearing	Water-insoluble, (________) dehydration and clearing with xylene or toluene
USE CASES	(_________) mounting for water-soluble stains (fat stains, PAS, metachromatic stain, immunofluorescent stain)	(_________) mounting for routine histopathology stains (H&E, Masson’s Trichome, Gram stain)
ADVANTAGES	● Prevents tissue (_________) ● Ideal for preserving (_________) stains ● Simple, (_________)	● Provides (________) ● (_____________) for better clarity ● Suitable for (_________)

DISADVANTAGES	● Not permanent; can (_______) ● May not provide (_______) for high-magnification imaging	● Requires (_______), which may cause tissue shrinkage ● Toxicity concerns due to (______)
EXAMPLES	● Water ● Glycerin ● Glycerin jelly ● Farrant’s medium ● Apathy’s medium ● Brun’s fluid	● Canada Balsam ● DPX ● XAM ● Clarite ● Eukitt - common in practice ● Permount, Clearmount
BEST FOR	(_______), immunofluorescence	(__________), permanent slides

OTHERS	(_____________) added, almost to the point of saturation to reduce ‘bleeding’ of cationic dyes	Divided into Natural and (______) (used for (_______), electron microscopy)

(1.33) (1.46) (Temporary medium) (Semi-permanent

medium) (years) (Moderately) (easily) (Not suitable) (Inexpensive) (Nonpoisonous) (Standard moutant) (microorganism) (Phosphate buffered glycerol) ()

AQUEOUS MEDIA

(MOUNTANT)

	COMPOSITION	GLYCERIN
R.I.	(_________)	(______)
USES	(_________)	Preservative action, (________) , Sets hard, Keep sections for (____) after ringing

REMARKS	● (_____) transparent ● Evaporates (______) ● for OIO	● (_______) ● (________) ● (_______) for fat stains ● Can be attacked by (_________) ● (________) - immunofluorescence

(high-volume labs) (small labs) ( large workloads) (backup method) (Automated Slide Labelers) (Diamond tipped pencil/leaded pencil) (● Barcode or QR code

● Patient’s name or Initial

● Accession Number

● Specimen Type (Liver Bx)

● Stain Used (H&E, PAS)

● Date of Processing) (● Patient’s Name or Initial

● Accession Number

● Specimen Type

● Stain Type

● Stain Used

● Date of Processing) () () () ()

	AUTOMATED SLIDE LABELING	MANUAL SLIDE LABELING
FLEXIBILITY	Best for (________) with standardized processes	Suitable for (_____) or when automation is unavailable
REDUNDANCY	Reliable for (________); reduces mix-ups	Acts as a (______) when machines fail

WHAT TO USE	(______) (Leica IP C, Sakura AutoWrite, Thermo Fisher PrintMate)	(__________): Ideally for frosted-end glass slides
WHAT TO INCLUDE

It depends on different laboratory hospital protocols as long as they are able to distinguish the slides to its corresponding patient’s tissue specimen.

(LABELING)

(● Patient’s name or initials

● Accession number

● Specimen Type

● Stain used

● Date of processing)

(_________)

● In histopathology, proper slide labeling is essential for

maintaining accurate records and ensuring that each

specimen is correctly identified

What to Include:

(ACCESSION NUMBERS) (surgical) (cytological) (Pap smear specimens) (S-25-1075)

● unique identifiers assigned to each specimen ● usually include the year the specimen was collected with varying prefixes for different types of specimens:

○ "S" = (______)

○ "C" = (_______)

○ "P" = (__________).

(__________) represent the 1,075th surgical

specimen received for the year 2025.

● If more than one tissue section slides received from one whole specimen of patient, addition of either alphabetical or numerical accession codes.

(GLYCERIN JELLY) (1.47) (fat stains) (viewing) (fade) (Polyvinyl alcohol) (FARRANT’S MEDIUM) (1.43) (fat stains) (harden) (Arsenic trioxide) (APATHY’S MEDIUM) (1.52) (methylene blue-stained) (CANADA BALSAM) (1.524) (whole mounts and thick sections) (age and oxidizes xylene) (Transparent and colorless) (w/o granulation) (DPX (Dibutyl Phthalate & Xylene) (1.532) (small tissue sections) (cleaned of excess mountant) (Dries rapidly)

AQUEOUS MEDIA

MOUNTANT	R.I. & USES	REMARKS
(______)	R.I.: (_____) USES: Standard mountant when dehydration and clearing with xylene are not performed (as in (_____) )	● Gelatin + Glycerol + Distilled water + Phenol crystals (preservative) ● Provide best (______) ● Stains tend to (____) ● Solidify on storage, does not set ● (______) - alternative for immunofluorescence
(_______)	R.I.: (_____) USES: Takes longer to harden and requires ringing for fat stains	● Gum arabic + Distilled water + Glycerol + Sodium merthiolate ● Does not solidify upon storage ● Longer time to (____) ● (______) - substitute ● Potassium acetate is added

(______)	R.I.: (________) USES: Used for (______________) nerve preparation; for fluorescent microscopy	● Glucose + Glycerin + Spirits of camphor + Distilled water
(______)	R.I.: (_______) USES: For (______________)	● Natural resin (Abus balsamea) ● Darkens with (________), making it acidic ● (________&______) ● Sets hard (________) ● Expensive
(______)	R.I.: (_______) USES: For (_______)	● Colorless, natural medium ● Stains are preserved ● Set quickly ● Greater advantage over CB as ● slides can be (___________) ● Produces shrinkage upon drying ● (____________)

(XAM) (1.52) (For permanent mounting) (Synthetic resin) (w/o retraction) (stain well) (Pale yellow) (CLARITE) (1.544) (For permanent mounting) (Synthetic resin) (60%)

NON-AQUEOUS MEDIA/RESINOUS

MOUNTANT	R.I. & USES	REMARKS
(_______)	R.I.: (_____) USES: (____)	● (______) mixture in xylene ● Dries quickly (_______) ● Preserve (______) ● (_______) or colorless
(_______)	R.I.: (______) USES: (_____)	● (__________) ● Diluted to (____) with xylene

(CB 1.524) (1.52) (1.51-1.52) (Water 1.33) (glass) () () () () () () () () () ()

IMPORTANCE OF MOUNTING MEDIA BASED ON REFRACTION

	Homogeneous (oil) immersion	Water immersion
RI	(________)	(_______)
REMARKS	closely matches the RI of glass slides (____) and objective lenses (_________)	Significantly lower RI than (_____), causing more light refraction, image distortion

(clean and flat surface) (consistency ) (sufficient drop) (capillary action)

STEPS ON COVER SLIPPING

1. Work on a (____________)

2. Check the (_________) of the mounting medium

3. Drop a (________) of mounting media (either

directly on the stained slide or on the coverslip)

4. Place the coverslip, and allow the mountant to

spread through (___________)

NOTES ON COVER SLIPPING

1. Do not allow the tissue to dry out during

coverslipping

Excess xylene + mountant could form bubbles

Too little amount of mountant may

Bubbles could be removed by gently pressing on

the coverslip using a mounting needle

If the bubble could not be removed, remount by

soaking it back to XYLENE

(IMMEDIATE EXAMINATION) (temporary)

(________________)

● Mount the broken slide onto a clean, xylene-moist

slide

● Add a drop of mounting media (Clarite or Permount)

● Use this temporary setup while a new section is being

cut and stained.

(xylene) (37°C incubator) (6 parts

butyl acetate and 1 part Durofix) (37°C incubator for 30) (sharp scalpel blade) (cold water) () () () () () () () () () () () () () () () () ()

TRANSFERRING AN IMPORTANT SECTION

● If replacement is unavailable:

○ Soak the broken slide in (_____).

○ Place the slide in a (________) until the

mountant is fully removed

○ Cover the slide with a mixture of (______________).

○ Leave in the (___________) minutes

until the mixture hardens into a film

○ Use (____________) to cut around the

section.

○ Place the slide in (______) to allow the film

and section to float off.

○ Mount the floating film onto a clean slide.