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In this article we will discuss about the application of some histochemical methods in the study of plants: 1. Procedure for Histochemical Localization of Total Insoluble Polysaccharides 2. Histochemical Localization of Starch by 1 Kl Method 3. Histochemical Localization of Proteins by Bromophenol Blue Method and few others.
1. Procedure for Histochemical Localization of Total Insoluble Polysaccharides:
1. Place the deparaffined plant tissue sections in 0.5% aqueous HIO4 for 10-15 min at room temp.
2. Wash in running water for 10 min.
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3. Place in Schiff’s reagent for 15-20 min (Schiff’s reagent is prepared by dissolving 0.5 g of basic function and 0.5 g of sodium meta-bisulfite in 100 ml 100 ml 0.15 N HCl. Shake the mixture for 2-3 hrs. Add 30 mg fresh decolorizing charcoal and shake for 5 min. Filter and keep the colourless solution in a coloured bottle in the refrigerator).
4. Rinse the sections in water and transfer them into 2% sodium bisulfite for 1-2 min.
5. Wash again in running tap water for 5-10 min.
6. Dehydrate and mount in DPX.
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Observation:
Intense purplish-red colour shows the presence of insoluble polysaccharides.
2. Histochemical Localization of Starch by 1 Kl Method:
1. Place the sections of the plant in 1 KI solution (dissolve 2 gm. of KI in 100 ml of water and then dissolve 0.2 gm. of iodine in the KI solution) for 10 min.
2. Wash in water and mount.
Observation:
The old starch appears blue to black in colour, whereas newly formed starch appears red to purple.
3. Histochemical Localization of Proteins by Bromophenol Blue Method:
1. Stain the section in 1% HgCl and 0.05% sodium bromophenol blue in 2% aqueous glacial acetic acid for 15 min at room temperature.
2. Rinse for 20 min in two changes of 9.5% aqueous acetic acid.
3. Blot the sections and dehydrate rapidly in two changes of ethanol.
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4. Clear in xylene and mount in DPX.
Observation:
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Proteins are stained blue.
4. Histochemical Localization of Total Lipids by Nile blue Procedures:
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1. Stain the sections with 1 % aqueous Nile blue at 50°C for 3-4 min.
2. Stain with 1% aqueous Nile blue at 50°C for 3-4 min.
3. Rinse in distilled water and immerse in warm 3% acetic acid solution for 30 sec.
4. Wash thoroughly in distilled water and mount.
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Observation:
Fats, oils and waxes (the neutral lipids) are stained red; the free fatty acids and phospholipids (the acidic lipids) are stained blue.
5. Histochemical Localization of Nucleic Acid by Azure B Method for DNA and RNA:
1. Place the tissue in 0.25 mg/mol solution of Azure B in citrate buffer at pH 4.0 for 2 hours at 50°C.
2. Wash in water and place in pure butanol for 30 min. Leave overnight if stained too deep in butanol.
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Observation:
DNA sites appear greenish-blue and RNA purple or dark blue.
6. Histochemical Localization of Total Phenols by Ferric Chloride Test:
1. Place the fresh section in 10% aqueous ferric chloride or 10% ferric chloride in 95% ethanol for 10 min or more.
2. Wash in water and mount.
Observation:
Materials containing phenols are stained bluish in colour as a result of the formation of complex iron salt.
