Stains

What stain is pictured here ?

Mayer Mucicarmine

Staining technique: Mayer Mucicarmine  

 

Principle of the technique:  Stains Carboxylated and Sulfonated mucins NOT neutral mucins, use for ID of adenocarcinomas  and certain fungal structures  

Purpose of stain: Staining of Epithelial mucin and demonstrates Cryptococcus neoformans  

Results:  

-Mucin Deep - rose to red  

-Capsule of Cryptococcus spp -  deep rose to red  

-Nuclei-  black 

-other tissue elements-  Blue or yellow  

Sources of error: Mucicarmine will gradually deteriorate over time, keep solution fresh and use good control to monitor for decrease in staining. Carminophilic properties will be obscured if sections are overstained with either hematoxylin or metanil yellow  

Preferred fixative: 10%NFB  

Preferred thickness: 4-5um  

Microscope used: light  

Control tissue: unautolyzed colon, small intestine or appendix  

Major reagents and purpose 

Major reagents	Purpose
Weigert Iron Hematoxylin	Nuclear Stain: An iron-lake progressive dye where ferric chloride acts as both the oxidizer and the mordant. This forms a robust iron-hematein complex that resists decolorization by the highly acidic mucicarmine solution applied immediately after.
Mucicarmine Stock Solution	Aluminum form chelation complex with carmine the resulting compound has net (+) charge and attaches to the acid groups of mucins
Metanil yellow	Counterstain anionic dye colors cytoplasm

What stain is pictured here?

Staining technique: Periodic acid-Schiff PAS  

Principle of the technique: Certain tissue components are oxidized into aldehydes by periodic acid. Schiff reagent is used to demonstrate these aldehydes.  

Purpose of stain: Demonstrate polysaccharides, neutral mucosubstances and basement membranes.  

Results: • PAS-Positive Entities (Magenta/Bright Pink): Glycogen, neutral mucins (goblet cells), basement membranes (renal glomeruli), fungal cell walls (chitin/glucan), and Tropheryma whipplei (the causative agent of Whipple's disease, found within macrophage lysosomes). 

• Nuclei: Blue (typically counterstained with Mayer's or Harris Hematoxylin). 

• Background: Light pink, green, or purple depending on whether a counterstain like hematoxylin or light green is used 

Preferred fixative:  10%NBF or Bouin Solution. Blood smears should be fixed in methyl alcohol 10-15mins.  

Preferred thickness: 4-5um kidney 1-2um  

Control tissue: kidney most sensitive control, also good for basement membranes but to detect glycogen use liver or sections of cervix include both ecto and endo.  

Major reagents	Purpose
Periodic acid	Oxidizing agent to aldehydes by periodic acid, reactive group is 1,2 – glycol group
Schiff Reagent	Colorless basic fuchsin
Potassium Metabisulfite	Rinse excess schiff

 

What stain is pictured here?

Staining technique: Alcian Blue 2.5 

Principle of the technique: Stains sulfated and carboxylated acid mucopolysaccharides and sulfated and carboxylated sialomucins or glycoprotein. Alcain blue forms salt linkages with acid groups of acid mucopolysaccharides  

Purpose of stain: Demonstration of acid mucopolysaccharides  

Results: weaky acidic sulfated mucosubstances, hyaluronic acid and sialomucins   - dark blue  

Background   - pink to red  

Nuclei – Red   

Preferred fixative: 10% NFB or Bouins Solution  

Preferred thickness: 4-5um  

 Control tissue: unautolyzed small intestine appendix or colon as positive control  

Major reagents	Purpose
Acetic Acid Solution	Chemical Conditioner / Buffer: Used as a pre-treatment to lower the tissue sections down to an exact pH of 2.5 before the dye arrives. This protects the upcoming alcian blue bath from water dilution and prevents pH fluctuations that would skew dye binding properties
Alcian Blue, pH 2.5 Dye	Primary Dye: A water-soluble, copper-phthalocyanine basic dye carrying a permanent positive charge. At pH 2.5, it reacts strictly via salt linkages (ionic bonds) with the ionized carboxylated and sulfated groups of acid mucopolysaccharides and sialomucins.
Nuclear Fast Red	Counterstain: A basic nuclear dye that utilizes an aluminum sulfate mordant to create a pink-red dye-lake complex. This targets DNA phosphate groups to provide high visual contrast against the bright blue target mucins.

What stain is pictured here?

Staining technique:  Congo red  

Principle of the technique:  Green bifringence following congo red staining most specific technique for demonstration of amyloid.  

Purpose of stain: Demonstration of amyloid in tissue  

Results:  

Amyloid – Deep pink to red  under light  apple green birefringence under polarized light  

Elastic tissue – Pale pink  

Nuclei – Blue 

Sources of error: Amyloid is a isomer of cellulose and resembles cellulose in chemical reactions. Prolonged storage in 10% formalin will cause gradual decrease in staining intensity  

Preferred fixative: Alcohol or Carnoy solution  

Preferred thickness: 8-10um 

Microscope used: light, polarized light microscope  

Control tissue: sections containing amyloid  newly formed deposits stain better  

Major reagents Purpose 

Major reagents	Purpose
Congo Red Staining Solution	Primary Dye:  Green birefringence Is intrinsic property of the amyloid fibril congo red complex and is the function of the parallel alignment of dye molecules and amyloid fibrils

 

 

 

Staining technique showed

Congo Red

Substance stained red in this image

Amyloid

Other techniques that may demonstrate amyloid ?

Thioflavin T, crystal violet