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Histological Identification of Lipids

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Histological Identification of Lipids Histological Identification of Lipids Dr. Kumari Aditi, Assistant Professor, PG Department of Zoology, MU Lipids fall in the category of heterogeneous biomolecules that are characterized by properties such as varying solubility in chemical solvents, presence/absence of charge etc. For histochemical detection of lipids in tissue samples, several chromogenic or colour producing dyes and stains are employed widely. We can visualize lipid deposits in histological sections using these specific dyes. Four lipid-specific dyes namely Sudan Black B, Sudan III, Sudan IV and Nile Blue Sulphate are described below. 1. Sudan Black B (SBB) Synonym: Solvent black 3, Fat Black HB SBB are fat-soluble diazo dye primarily used for the staining of neutral lipids and triglycerides. SBB is a member of azobenzenes, a bis(azo) compound and a member of perimidines (Figure 1). This stain is also used for the visualization of a wide variety of lipids such as phospholipids and intracellular lipids such as sterols. Black Sudan B is not lipid specific like other Sudan dyes and can also be used for chromosome staining, visualizing Golgi apparatus and leukocyte granules. The dyes of the Sudan family form a group of solvent-soluble lipid dyes also known as lysochromes or lipid-soluble dyes. SBB is slightly basic in nature, so it will easily bind to acidic groups in lipids and can also stain phospholipids. Staining pattern of SBB is very similar to myeloperoxidase reaction, with better staining of granulocytes such as eosinophils and faint staining of monocytes. SBB does not stain B or T lymphocytes. SBB is effectively used in the detection of hematological pathologies. SBB is highly sensitive and specific for lipid staining. It can also stain lipids in formalin fixed tissue sections; however, it is not suitable for paraffin embedded 1 tissue sections. Sudan Black B stains phospholipids Gray and Neutral fats Blue- black (Figure 2). Cell nucleus can be stained in red colour using Nuclear Fast Red when using SBB. Source: https://pubchem.ncbi.nlm.nih.gov/compound/Sudan-black-B Figure 1: Chemical structure of Sudan Black B Dye Source: https://psl-equip.com/product/fat-stain-kit-sudan-black-b-250-ml/ Figure 2: Fat staining by Sudan Black B (arrowheads) 2 2. Sudan III Synonyms: Solvent red 23, Cerasin Red Sudan III is a member of azobenzenes, a bis(azo) compound and a member of naphthols (Figure 3). It is also a lysochrome or lipid-soluble dye predominantly used for demonstrating triglycerides in frozen tissue sections. Exceptionally, Sudan III may also stain some protein bound lipids in paraffin sections. Sudan III acts as a fluorochrome besides a histological lipid-specific dye. It also acts as a carcinogen. For use in histochemistry, a saturated solution of SUDAN III in isopropanol is prepared and tissue is counterstained in acid alum hematoxylin (approx. 0.1%) for 5 min. Lipids appear yellow-orange in colour upon staining with Sudan III (Figure 4). Nuclei will appear blue and cytoplasm will appear green. Since lysochrome dyes like SUDAN III are soluble in non-polar solvents, including xylene and toluene, and also in ethanol, these solvents must not be applied to the sections after lipids have been stained or the dye will dissolve out. Source: https://pubchem.ncbi.nlm.nih.gov/compound/Sudan-III#section=Structures Figure 3: Chemical structure of Sudan III dye 3 Figure 4: Sudan III staining of triglycerides 3. Sudan IV Synonyms: Solvent Red 24, Scarlet Red Sudan IV is a red β-naphthol disazo dye. Its basic structure is same as Sudan III with two extra methyl groups (Figure 5). Because of these groups, Sudan IV is deeper and more intense in color (Figure 6). With hydroxyl group present at ortho position, it has similar physical properties to Sudan III and is soluble in lipid. Sudan IV is also a lysochrome dye used widely for staining triglycerides in frozen tissue sections. The colour imparted by Sudan IV is because of the solubility of dye in lipids and it does not involve any chemical bonding. Sudan IV may also stain some protein bound lipids in paraffin sections. Sudan IV has been used in industries to colour oils, wax, shoe-polish, wood stains etc. Sudan IV also acts as a carcinogen. For histological staining, a saturated solution of Sudan IV is prepared and used in tissues. Source: http://www.chemspider.com/ImageView.aspx?id=11252033 Figure 5: Chemical structure of Sudan IV dye 4 Source: Porzionato, A. et al, 2013 Figure 6: SUDAN IV staining of fats in tissue (orange-red) 4. Nile Blue Sulphate Synonym: Nile Blue sulfate, Nile Blue A sulfate Nile blue sulphate is a basic oxazine dye (Figure 7) and is useful for staining fats. For dye preparation, 1% acid-hydrolysed Nile blue is prepared by boiling 200 ml of Nile Blue Sulphate with 10 ml of 1% H2SO4 for 4 hr with heating at controlled temperature. Nile blue sulfate can be used for staining both tissue lipid and extracted lipid. It is also used to distinguish melanins and lipofuscins. Nile blue sulphate is soluble in water and ethyl alcohol. It can also stain PHB (Poly-p3-hydroxybutyrate) granules in bacteria. PHB granules display bright orange fluorescence upon staining with Nile blue Sulfate. The dye is quite stable and does not fade easily from cells by decolouring procedures. Polar lipids such as phospholipids are coloured intense blue by the oxazine base of the dye whereas neutral lipids such as triglycerides, steroids and cholesteryl esters are stained red by its oxazone derivative. Free fatty acids are coloured Magneta-red due to a blend of red (oxazone) and blue (oxazine) staining. Aqueous solution of nile blue sulphate contains blue cation of the dye and also forms fluorescent red oxazone (Nile Red) by oxidation and a non- 5 fluorescent orange-red imino base. So, we can clearly distinguish phospholipids from the other main lipid classes in tissue sections by using nile blue sulphate dye. Source: https://pubchem.ncbi.nlm.nih.gov/compound/Nile-Blue-A-sulfate#section=2D-Structure Figure 7: Chemical structure of Nile Blue Sulphate *** 6 .
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