Stains for Retic, Muscle, Basement Membranes, and Lipids

What are reticular fibers?

Collagen fibers not demonstrated by other stains

What is the purpose of the Gomori stain for reticular fibers?

To demonstrate reticular fibers or monitor change in normal reticular pattern

What is oxidized in the Gomori stain for reticular fibers?

Hexose sugars in reticulin

What is the oxidizer in the Gomori stain for reticular fibers?

Potassium permanganate

What are the hexose sugars oxidized to in the Gomori stain for reticular fibers?

Aldehydes

How is excess potasium permanganate removed in the Gomori stain for reticular fibers?

Potassium metabisulfite

What is the sensitizer in the Gomori stain for reticular fibers?

Ferric ammonium sulfate

What replaces ferric ammonium sulfate in the Gomori stain for reticular fibers?

Silver from diamine silver solution (impregnation)

What reduces silver to metallic visible form in Gomori stain for reticular fibers?

Formalin

What tones the visible silver in the Gomori stain for reticular fibers?

Gold chloride

What removes excess unreacted silver in the Gomori stain for reticular fibers?

Sodium thiosulfate

What is the fixative used for the Gomori stain for reticular fibers?

NBF

What is the best control for the Gomori stain for reticular fibers?

Liver

What is stained black by the Gomori stain for reticular fibers?

Reticulin

What is stained light brown by the Gomori stain for reticular fibers?

All other collagen

What is the purpose of the Gordon and Sweets stain?

To demonstrate reticulin

What is the main difference between the Gomori stain and the Gordon and Sweets stain for reticular fibers?

Oxalic acid removes oxidizer (potassium permanganate) instead of potassium metabisulfite

What is a benefit of the Gordon and Sweets stain?

Reduces background staining

What stains black in the Gordon and Sweets stain?

Reticulin

What is the purpose of the Mallory PTAH stain?

Demonstrates cross striations in muscle for diagnosis of rhabdomyosarcomas

What is PTAH

Phosphotungstic acid hematoxylin

What are rhabdomyosarcomas?

Striated muscle tumors

What type of solution is the Mallory PTAH?

Polychromatic

What will happen if slides are not rapidly dehydrated after Mallory PTAH stain?

Red-brown color of collagen is lost

How much phosphotungstic acid is in solution compared to hematein in Mallory PTAH stain?

20:1 (Much more)

What is the mechanism for the Mallory PTAH stain?

Free tungsten ions bind to all available hematein to produce a blue lake, remaining phosphotungstic acid dyes red-brown

What is dyed blue in the Mallory PTAH stain?

Cross striations in muscle, fibrin, nuclei

What is dyed red-brown in the Mallory PTAH stain?

Collagen

What fixative is preferred for the Mallory PTAH stain?

Zenker, but NBF is okay

Can you do the Mallory PTAH without using mercuric solutions and fixatives?

Yes, acidic dichromate and acidic potassium permanganate is used

What is the best stain for the demonstration of basement membranes?

Periodic acid methenamine silver (microwave)

What is the periodic acid methenamine silver stain typically used for?

To demonstrate the basement membranes of kidney to identify glomerular abnormalities

What is the purpose of periodic acid in the periodic acid methenamine silver stain?

Oxidizes the carbohydrates found in the basement membrane to aldehydes

What is the purpose of methenamine silver in the periodic acid methenamine silver stain?

Silver ions from this solution bind to aldehydes ad are reduced to metallic form

What gives the periodic acid methenamine silver stain the proper alkalinity for the reaction to occur?

Methenamine

What is the buffer in the periodic acid methenamine silver stain?

Sodium borate

What is the preferred fixative for the periodic acid methenamine silver stain? Which should be avoided?

NBF, mercuric fixatives need to be avoided

What is the control for the periodic acid methenamine silver stain?

Kidney sections cut at 1-2 um

What is the counterstain of the periodic acid methenamine silver stain?

Light green

What stains black with the periodic acid methenamine silver stain?

Basement membranes, especially in kidney

What stains green with the periodic acid methenamine silver stain?

Background tissues

What other stain can stain basement membranes other than the periodic acid methenamine silver stain?

PAS

What is the purpose of the Oil red O stain?

Demonstration of neutral lipids in frozen tissue, liposarcomas

Can the Oil red O stain be used on paraffin sections?

No, must be frozen because dehydrating and clearing agents dissolve fat

What type of stain is the Oil red O?

Physical

Why is the Oil red O stain a physical stain?

Dye is more soluble in fats than in solvent, so it moves into the fats

What are the two best solvents for the Oil red O method?

Propylene glycol and isopropanol

What is the preferred fixative for the Oil red O method?

NBF or calcium formol

What fixatives must be avoided during the Oil red O method?

Alcohols

What thickness are sections cut at for the Oil red O method?

10 um

What is the section stained with after Oil red O?

Hams acetic acid hematoxylin, blued with ammonia

What are important coverslip techniques for the Oil red O method?

Use aqueous mounting media and do not press hard on coverslip

Is there a control associated with the Oil red O method?

No

What color does fat stain with the Oil red O method?

Red

Is there a way to stain fat that needs to be embedded in paraffin?

Yes, osmium tetroxide

What is the mechanism of osmium tetroxide for fat staining?

Osmium tetroxide chemically combines with fat, blackening it and making it insoluble in alcohols and xylenes

What is the fixative for the osmium tetroxide for fat staining?

NBF

What is used to differentiate in the osmium tetroxide stain for fat?

Periodic acid

What is special about the timing of the osmium tetroxide stain for fat?

It is done before processing

What can be used as a counterstain for fat stained with osmium tetroxide?

Trichrome

What are the staining results for osmium tetroxide fat stain?

Fat is black, cytoplasm is gray

What important microtomy techniques should be considered with osmium tetroxide stain for fat?

Osmium tetroxide has low penetrating power so the first few sections should be taken from block

What important grossing techniques should be considered with osmium tetroxide stain for fat?

Osmium tetroxide has low penetrating power so gross sections should be cut at a thickness no greater than 2mm before staining