Routine Staining Flashcards by Lori Hay

What technique uses light green to stain collagen?

Masson’s Trichrome

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Which technique uses Iodine as a trapping agent

Verhoeff’s Van Gieson

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What is the purpose of taking slide down to water and how is it done in general?

Take slides to water (2 min per station). Purpose: Remove any residual wax around and in tissue before staining. Needs to be removed as most dyes are aqueous solutions and do not mix with wax.
a. Deparaffinization – using xylene
b. Removal of xylene – using 100% ethanol.
c. Re-hydration – using descending grades of ethanol.

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What happens if the paraffin is not completely removed?

Stain will not penetrate tisse.

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What happen if xylene is not completely removed during the “Take Down to Water”?

The 95% and 70% alcohol solutions will turn cloudy, indicating contamination. This is something to watch out for in the lab.

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What is the reason for using descending % of alcohols in taking down to water|?

Descending levels of alcohols are used in the “Take Down to Water” to prevent tissue from falling off the slide.

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What is the process we use to “Take Down to Water” in the lab?

The slides are dipped in each solution for two minutes each in the following: 
Xylene - 2 dishes
100% Ethanol - 2 dishes
95% Ethanol - 1 dish
70% Ethanol - 1 dish
Water (tap or distilled) - 1 dish.

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What is the purpose of the Dehydrate in Dehydrate-Clear-Mount?

Dehydration
• Removes water using increasing % conc. of alcohol (don’t leave slides too long in alcohols as it can remove stain from slides)

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What is the purpose of clearing in DCM?

Clearing
•	Many staining dishes of xylene used
•	Raises RI (Refractive index)
•	Miscible with permount
•	Slides may be left in xylene if mounting must be done later due to workload.

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What is the purpose of coverslipping in DCM?

Mount – Coverslipping
• Provides protection on tissue and allows for long-term storage.
• Does not support growth of molds and bacteria.

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What type of mounting medium is hydrophobic and the most routinely used?

Permount, synthetic resin with a refraction index of 1.52.

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What is the size of a coverslip?

22 x 22 mm or 22 x 50 mm and we use No. 1 - 0.15 mm thick.

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What type of permount is hydrophilic and what is it suitable for?

Aquamount
It is suitable for lipids, enzymes, metachromatic stains.
RI of 1.42

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What are characteristics of a good mounting medium when coverslipping (try to list 5)?

• transparent and nearly colorless
• flows easily
• releases air bubbles
• does not support the growth of molds or bacteria
• does not dissolve or corrode tissue constituents to be
studied
• dries to non-stickiness within a reasonably short period
of time
• refractive index close to that of glass and tissue (1.518-
1.530)
• fills tiniest spaces present within cells and tissues
• does not or appear granular when set
• does not fade, stain or bleed stain

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What may be the reason if we get bubbles under the coverslip?

Mounting media aspirated too vigorously from the bottle before application to the slide. (be gentler)
Mounting media (permount) too thick. (use fresh, and/or thin with xylene)
Too little mounting medium used in coverslipping technique. (use bigger drop)
Mounting medium spreads too fast between the slide and coverslip. (more hand control)

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What may be the reason if we find excess mounting medium on the top of the coverslip?

Excessive amount of mounting medium used (use less).

What may be the reason if we see rainbow colours on the coverslip?

Two coverslips are stuck together (gently pull coverslips apart).

What is hematoxylin? Does it have a colour?

Natural dye from the heartwood of the Logwood Tree.

No, it does not have a colour; it is in reduced form with no chromophore. Needs to be oxidized (also called ripening) to obtain a chromophore.

What are the two methods to oxidize (ripen) hematoxylin to hematein?

Chemical Method:
Na Iodate (Sodium Iodate),
HgCl (Mercuric Oxide, Mercuric Chloride),
FeCL3 (Ferric Chloride)

Natural Method: Allow hematoxylin to ripen-exposure to air and sunlight (6-8 weeks)

What is the purpose of oxidizing hematoxylin?

Purpose of oxidizing Hematoxylin:

To give the dye a chromophore and converts hematoxylin to hematein. Contains the Chromophore structure Quinoid.

What else does hematoxylin (hematin) need after oxidizing to stain the tissue?

A mordant is required.

Aluminum:
-Ammonium aluminum Sulfate, -Potassium aluminum Sulfate

Iron: Ferric Chloride

Lake =
BLUE - e.g. KAlSO4, NH4AlSO4
BLACK – e.g. FeCl3

What are the different types of Alum(inum) Hematoxylins?

Harris: P or R
Mayer’s: P or R (mainly P)
Ehrlich’s: R only
P = progressive, R = regressive

Ingredients are oxidizers, stabilizers/preservatives, and acidifying agents.

When are Iron Hematoxylins used? Role of Iron? Stability?

Used when strong acids follow nuclear stain (as the strong acid does not remove the iron hematoxylin).
Fe has dual role: Mordant and oxidizer.
No stabilizer –> over-oxidized (means double bond gets lost)

Must be made fresh!

Name some examples of Iron Hematoxylins?

Weigert’s: Progressive, short lived (2-3 hours)

Verhoeff’s:
Regressive, short lived (<24 hours)

How does an H&E obtain black nuclei?

Black lakes will stain the nuclei of cells a black color using a mixture of hematein with ferric salts (mordent) --> Iron Hematoxylins.

What are the main steps in the H&E stain technique?

1. Hematoxylin --> Nuclear Stain 2. Differentiating Agent --> Acid Alcohol 3. Blueing Agent --> Ammonia Water 4. Eosin --> Counterstain

Describe what "correct differentiation", "over differentiation" and "Under differentiation" means with the H&E technique in terms of what the slide looks like after differentiation?

Correct Differentiation: The dye lake remains attached to the phosphate groups but not on the other groups. Nuclei are red, other tissue components have no color. Over Differentiation: The dye lake is removed from the phosphate groups as well as the other acidic groups. Nuclei are pale staining or not colored, other tissue components have no color. Under Differentiation: The dye lake is not sufficiently removed from the sulphate or carboxyl groups. Nuclei are red and all other tissue components are pink.

What does hematoxylin and eosin both have in common?

A chromophore that is quinoid in shape.

What are the two different dye powders that can be used to make up Eosin dye solution?

Eosin Y and Phloxine B. Actual products used depend on availability and pathologist preference.

What could be the problem if after staining you notice the nuclei are very pale (almost colourless)?

Could be any of the following reasons: 1. Over-differentiation --> too long in the acid alcohol. 2. Non long enough in the hematoxylin. 3. Hematoxylin is getting old.

What may be the problem if your nuclei are red-brown colour instead of blue?

Forgot to put slides in the ammonia water or it is becoming too weak.

What may be the problem if you have a pale counterstain background?

1. Not all the ammonia water has been removed. Ensure proper rinse to neutralize the alkaline pH before going into Eosin. 2. Not long enough in the eosin. 3. Eosin getting too old. Need to make fresh. 4. Dehydrating through alcohols too slow, i.e. in the DCM (as they can remove the stain).

What could be the problem if you are getting bluish colour on tissue where it the eosin (red) should be?

It may be under differentiated in acid alcohol, so it didn't remove the hematoxylin on the tissue where you don't want hematoxylin to be. Solution: Dip it more in the acid alcohol before going on.

What is the purpose of rapid H&E and the brief list of the main steps?

Purpose is to provide a diagnostic service to a surgeon during an operation. 1. Frozen sectioning. 2. Brief fixation. 3. H&E < 5 mins. Similar to regular H&E just shortened times in each reagent. Stain quality is not as good but gives decent results for diagnosis. More detailed procedure is in the notes.