Histology Stains | BME 320

Hematoxylin & Eosin

cancer, purple/blue

PAS

carbs, purple

Safranin-O

cartilage & extra cellular proteins, orange/red

Fast Green

counterstain for safranin-O, noncartilage (green), nuclei (purple).

Giemsa

blood smears, allergic/immune response (WBC), parasites, blue/purple

Masson’s Trichrome

differentiates b/w collagen (blue), muscle (red), cytoplasm (red), nuclei (black), muscles, cardiac, liver, kidney tissue

Verhoeff-Van Giesson

elastic tissue (blue/black), collagen (red), elastofibirosis, defective scaffold for elastic decomposition (Marfan Syndrome)

Sudan Black SBB

fats (blue/black), differentiating myeloid (has fats) vs lymphoid (doesn’t have fats) leukemia.

Alcian Blue

sugars (blue), Barrett’s esophagus, bronchial asthma, bronchiectasis, cystic fibrosis

Kulver - Barrera

neuron nuclei (purple), myelin sheath (blue)

A researcher suspects a new chemical called TUMOR-X increases abnormal tissue growth in rat kidney. The researcher treats rats for 6 weeks and wants to examine whether the tissue shows signs consistent with cancer-like changes.

a. Describe your experimental groups (4 pts)

b. What stain is most appropriate for this question and why? (4 pts)

c. Describe what you anticipate your stained tissue will look like in control vs treated animals (4 pts)

Stain: Hematoxylin & Eosin (H&E)
Why: General tissue morphology + commonly used to identify cancer/abnormal tissue structure
Colors: Mostly purple/blue (nuclei stain strongly)

A researcher is studying a metabolic disorder in rat liver. The disorder is suspected to cause abnormal accumulation of carbohydrates in the tissue. They want to test a drug called CARB-BLOCK.

a. Describe your experimental groups (4 pts)

b. What stain is most appropriate and why? (4 pts)

c. Describe expected appearance in control vs treated (4 pts)

Stain: PAS
Why: Confirms presence of carbohydrates
Color: Purple

A researcher is studying joint degeneration in rats. The disease causes damage to cartilage over time. They want to test a new drug called JOINT-SAVE that is expected to protect cartilage.

a. Describe your experimental groups (4 pts)

b. What stain is most appropriate and why? (4 pts)

c. Describe what the tissue should look like if the drug works vs doesn’t (4 pts)

Stain: Safranin-O + Fast Green
Why: Identifies cartilage and extracellular proteins
Colors:

• Cartilage = orange/red

• Non-cartilage = green

• Nuclei = purple

A researcher suspects a new drug called ALLER-STOP reduces allergic immune responses in rats. They collect blood samples and prepare blood smears. They also want to be able to detect if parasites are present.

a. Describe your experimental groups (4 pts)

b. What stain is most appropriate and why? (4 pts)

c. Describe what you anticipate will change in treated vs untreated samples (4 pts)

Stain: Giemsa
Why: Used on blood smears only, detects immune/allergic response patterns + parasites
Colors: Your professor may not require exact cell colors, but generally it differentiates blood cell types.

A researcher is studying a disease affecting cardiac tissue and suspects it causes increased deposition of collagen in the heart. They test a new drug called CARDIO-GUARD.

a. Describe your experimental groups (4 pts)

b. What stain is most appropriate and why? (4 pts)

c. Describe expected colors/appearance in control vs treated (4 pts)

Stain: Masson’s Trichrome
Why: Differentiates collagen vs muscle (great for heart, liver, kidney)
Colors:

• Collagen = blue

• Muscle/cytoplasm = red

• Nuclei = black

A researcher is studying a disorder affecting blood vessels. The disorder causes weakening of vessel walls due to problems with the elastic scaffold (similar to what happens in Marfan syndrome). They test a drug called ELASTA-FIX.

a. Describe your experimental groups (4 pts)

b. What stain is most appropriate and why? (4 pts)

c. Describe what the results should look like (colors) if the drug works (4 pts)

Stain: Verhoeff–Van Gieson (VVG)
Why: Highlights elastic tissue scaffold + collagen; used for vessels/skin/lungs/ligaments
Colors:

• Elastic tissue = blue/black

• Collagen = red

A researcher is studying a blood disorder in rats and suspects the disease is acute myeloid leukemia (AML) rather than lymphoid leukemia. They also suspect the condition is associated with increased cellular senescence. They want to stain for fats in the cells.

a. Describe your experimental groups (4 pts)

b. What stain is most appropriate and why? (4 pts)

c. Describe what the stained results would look like (colors) in AML vs non-AML samples (4 pts)

Stain: Sudan Black (SBB)
Why: Stains fats; used for AML vs lymphoid leukemia; detects senescence
Color: blue/black

A researcher is studying a disease affecting the airways and suspects it causes increased mucus production and accumulation of sugars. They want to test a drug called MUCUS-OFF.

a. Describe your experimental groups (4 pts)

b. What stain is most appropriate and why? (4 pts)

c. Describe what you expect the tissue to look like (colors) in untreated vs treated (4 pts)

Stain: Alcian Blue
Why: Stains sugars/mucus; used in airway mucus conditions + Barrett’s esophagus
Color: (Expect blue staining of mucus/sugars)

A researcher is studying a neurodegenerative disease in rat brain that causes damage to myelin sheaths. They test a drug called MYELIN-SAVE.

a. Describe your experimental groups (4 pts)

b. What stain is most appropriate and why? (4 pts)

c. Describe what you expect the tissue to look like (colors) if the drug works (4 pts)

Stain: Kluver–Barrera
Why: Identifies myelin + neuron nuclei
Colors:

• Myelin sheaths = blue

• Neuron nuclei = purple

A researcher is studying a disease in rat lungs. The airway lining is producing abnormally thick secretions, and the researcher suspects an increase in sugar-rich molecules in the airway.

They test a drug called AIR-DRY that should reduce the buildup.

a. Experimental groups (4 pts)

b. What stain is most appropriate and why? (4 pts)

c. What would you expect the tissue to look like in untreated vs treated? (4 pts)

Alcian Blue (mucus/sugars; airway diseases like asthma/CF)
Expected: more blue staining in untreated, less in treated.

A researcher is studying rat skin. The skin is becoming less stretchy over time, and the researcher suspects a breakdown in the tissue scaffold that normally provides elasticity.

They test a drug called SKIN-FLEX.

a. Experimental groups (4 pts)

b. What stain is most appropriate and why? (4 pts)

c. Describe expected colors/results if the drug works (4 pts)

Verhoeff–Van Gieson (VVG) (elastic scaffold breakdown)
Expected: elastic tissue blue/black; collagen red; diseased has weaker elastic signal

A researcher is studying rat kidney tissue. They suspect the disease causes abnormal tissue organization and changes consistent with uncontrolled growth.

They test a drug called CELL-CHECK.

a. Experimental groups (4 pts)

b. What stain is most appropriate and why? (4 pts)

c. Describe expected appearance + colors (4 pts)

H&E (general tissue organization + cancer-like changes)
Expected: purple/blue tissue features; abnormal architecture in untreated.

A researcher is studying rat liver. They suspect the tissue is accumulating too much stored energy in the form of carbohydrate-rich molecules.

They test a drug called GLYCO-STOP.

a. Experimental groups (4 pts)

b. What stain is most appropriate and why? (4 pts)

c. Expected colors/results in untreated vs treated (4 pts)

PAS (carbohydrates)
Expected: purple where carbs are; treated should show reduced purple.

A researcher is studying a joint disease in rats. The disease affects the smooth tissue that covers the ends of bones, and the researcher suspects it is wearing away.

They test a drug called JOINT-SHIELD.

a. Experimental groups (4 pts)

b. What stain is most appropriate and why? (4 pts)

c. Expected colors/results if the drug works vs fails (4 pts)

Safranin-O + Fast Green (cartilage)
Expected: cartilage orange/red; non-cartilage green; nuclei purple.

A researcher is studying rat heart tissue. The researcher suspects the disease increases stiff supportive tissue between muscle fibers, making the heart less efficient.

They test a drug called CARDIO-SOFT.

a. Experimental groups (4 pts)

b. What stain is most appropriate and why? (4 pts)

c. Describe expected colors/results (4 pts)

Masson’s Trichrome (collagen vs muscle in heart)
Expected: collagen blue, muscle/cytoplasm red, nuclei black.

A researcher is studying rat brain tissue. The disease causes a decline in nerve signal transmission, and the researcher suspects damage to the insulating structures around neurons.

They test a drug called NEURO-SAVE.

a. Experimental groups (4 pts)

b. What stain is most appropriate and why? (4 pts)

c. Describe expected appearance + colors (4 pts)

Kluver–Barrera (myelin + neuron nuclei)
Expected: myelin blue; neuron nuclei purple; damaged myelin = less blue.

A researcher suspects a rat population has been infected with a parasite. They want to evaluate whether infection levels drop after treatment with a drug called PARA-KILL.

The samples are collected as blood smears.

a. Experimental groups (4 pts)

b. What stain is most appropriate and why? (4 pts)

c. What changes would you look for in treated vs untreated? (4 pts)

Giemsa (blood smear, immune response, parasites)
Expected: parasite presence + altered blood cell patterns; treated should show fewer.

A researcher is studying rat blood samples and suspects a leukemia type involving cells with high fat/granule content. They want to distinguish between two leukemia types.

They test a drug called LEUKO-FIX.

a. Experimental groups (4 pts)

b. What stain is most appropriate and why? (4 pts)

c. Describe expected staining + colors (4 pts)

Sudan Black SBB (fats; AML vs lymphoid leukemia; senescence)
Expected: fats blue/black; AML more positive

A researcher is studying rat esophagus tissue and suspects the lining is producing abnormal sugar-containing material. They want to confirm the presence of these molecules.

They test a drug called ESO-CLEAR.

a. Experimental groups (4 pts)

b. What stain is most appropriate and why? (4 pts)

c. Describe expected colors/results (4 pts)

PAS or Alcian Blue depending on how she frames it

• If she emphasizes carbs generally → PAS (purple)

• If she emphasizes mucus/sugars like Barrett’s → Alcian Blue (blue)

What information should be in experimental groups

age, animal, the types of groups, dosage, timelines, amount of dosage, how dose is administered.

How do you prep the tissue?

1. put rat in gas chamber & kill it (CO2)

2. Perform cardiac puncture

3. First profuse w/ PBS while blood drains out

4. Then profuse w/ 4% PFA

5. Extract tissue of interest

6. Immerse the tissue in 10% PFA for a specific time depending on the sample.

When do you use OCT vs Paraffin?

Paraffin = best structure (candle wax mold, microtome)

OCT = best preservation of fats + fastest (gel + freeze, cryostat)

Describe the OCT procedure

1. expose sample to OCT compound

2. Samples are infused with OCT in a Cryomold then frozen

3. Samples can be sectioned into 5ul using a cryostat

Describe the paraffin procedure

1. Paraffin is melted @ 58C

2. Tissue is inserted into embedding cassettes

3. Cassettes w/ tissue undergo dehydration

4. Cassettes are transferred to the beaker w/ the melted paraffin + put back in the oven @ 58C for 45 min

5. Add samples to the casette container. Use warm tweezers to push tissue to the bottom of the mold. Let it stiffen on a cold plate. Add lid before it stiffens,

6. Expose tissue surface w/ rotary microtome & sharp blades, 4-5um sections.

Describe a general staining procedure

1. Rinse the tissue in water/buffer

2. Apply the stain

3. Rinse the stain with water

4. Rinse the stain with alcohol

What could go wrong

• the sample wasn’t kept warm while the tissue was extracted and prepped

• left in 10% PFA too long while prepping = poor dehydration = bad anatomical structures

• Butchered from sectioning

• Not fully dehydrated during prep

• dull blade during sectioning

• wash too much of the dye off

• the sections weren’t observed at the same microscope setting

What does OCT stand for?

Optimal Cutting Temperature

What should N be?

6-8

What’s a negative control group

no heart attack

What’s a positive control group?

heart attack

What’s the test group

heart attack w/ treatment

When making sections how big should they be?

10um

How do you quantify the data?

You’re looking at the area of the cells of interest and comparing one section to other sections

How many sections do you need?

You need at least 3 sections for each animal for each stain.

What things do you compare after the stains are done

The changes pre and post an event/treatment