Staining of proteins, enzymes and nucleic acids

• Simple proteins

  hydrolysis they yield only the amino acids and occasional small carbohydrate compounds
• Conjugated proteins

  simple proteins combined with some non-protein material in the body to form complex proteins
• Derived proteins

  proteins derived from simple or conjugated proteins by physical or chemical means.
• Most proteins in the cytoplasm are basic because they are positively charged due to the arginine and lysine amino acid residues.
• Therefore, eosin binds to these amino acids/proteins and stains them pink.
• Basophilic dyes are attracted to basic substances, which are cationic (positively charged) at physiologic pH.
• Fast Green


  acid dye that stains basic groups in the tissues, particularly basic protamines and histones which have higher isoelectrical points than the pH of the staining solution.
• Trichloracetic acid


  used to remove nucleic acid which would otherwise mask the basic group of protamines and histones.
• Peracetic Acid

  oxidizes cystine and cysteine, forming strong cysteic acid which is stained blue-green by a basic dye
• Sakaguchi’s test

  for arginine uses NaOH, sodium hypochlorite (Milton's reagent) and pyridine chloroform, producing orange-red color on objects containing arginine.
• Proteoglycans

  proteins that are heavily glycosylated
• Alcian Blue-PAS Staining for Proteoglycans

  by first staining all the acidic mucins with Alcian blue, those remaining acidic mucins which are also PAS positive will be chemically blocked and will not react further during the technique
• Feulgen technique


  demonstrates sugar
• Methyl green pyronin technique

  demonstrates phosphate
• Gallocyanin-chrome alum method


  demonstrates both DNA and RNA
• Feulgen Staining for Nuclear DNA

  used to identify chromosomal material or DNA in cell specimens

  RNA is not hydrolyzed by the HCl treatment and, thus, the reaction is DNA-specific
• Feulgen Staining for Nuclear DNA

  DNA - red purple

  Cytoplasm - green
• Methyl Green-Pyronin method for RNA and DNA

  DNA (Chromatin) Green or blue-green

  RNA (Nucleoli) Rose-red;

  Granules Dark rose-red

  Plasma cell cytoplasm Purple
• Fluorescein


  most widely used fluorochrome

  characteristic apple green emission is rarely seen as "auto fluorescence" in mammalian tissue, which is often blue in color
• Rhodamine


  conjugates absorb maximally in green light, exhibiting an orange-red emission, and are commonly used in two-color techniques
• Acridine Orange


  most commonly used fluorochrome to demonstrate DNA and RNA in fresh or fixed tissues, combining with nucleic acids in cells by salt linkages and cohesion
• Acriflavine


  an be used as an alternative to basic fuchsin in Schiff's reagent, for the Feulgen technique of acid hydrolysis.
• Coomassie Stains


  most popular anionic protein dye
• Ethylene Bromide


  sensitive, easy stain for DNA
• Silver Stains

  offer the highest sensitivity, although protocols are often time-consuming, complex, and do not offer sufficient reproducibility for quantitative analysis
• Fluorescent Stains

  ideal for protein study but are more expensive than Coomassie or silver stains and require either a CCD (charge-coupled device) camera or fluorescence scanner for gel imaging