Histochemical Staining of Plant Tissues

Histochemistry is a subfield of histology that focuses on identifying the chemical components of cells and tissues. Starch deposition occurs throughout the plant body, but seeds, the parenchyma of secondary vascular tissues in the stem and root, tubers, rhizomes, and maize are notably affected. The protoplast's main ergastic components are starch and proteins. Tannin, a diverse collection of phenol derivatives that are usually related to glucosides, is prevalent in the leaves (xylem) of many plants. Saponins are a type of saponin that is extremely rare. Fats are broadly dispersed throughout the plant body, and they are likely present in minute quantities in each plant cell. Fats are a common reserve resource in meristematic cells such as seeds, spores, and embryos. The breakdown product of carbohydrates is glucosides. Alkaloids are the byproducts of protein breakdown. Many plants have secondary products that are medicinally valuable. Using stains, markers, and light and electron microscopy, histochemistry studies the identification and distribution of chemical substances within and between biological cells. Histochemistry

to see, so place the tube containing the sections on a test tube rack and put aside for 2 minutes at room temperature.Use a 1 ml pipette to draw out 700 µl of 0.02% toluidine blue O solution. Add 700 µl of distilled water to rinse out the toluidine blue O solution. Repeat 3-4x or until the wash solution is clear. Use a 1 ml pipette with the pipette 1. Draw out 700 l of 0.02 percent toluidine blue O solution with a 1 ml pipette. Rinse away the toluidine blue O solution with 700 l of distilled water. Rep 3-4 times more, or until the wash solution is clear. Use a 1 ml pipette with a pipette tip cut so that the sections may be pipetted out easily without being damaged. 2. Pipette portions onto the tip of the pipette and onto a microscope slide, then cover with a coverslip. Under bright-field lighting, examine the portions.  Detection of Hydrophilic substances : Mucilage: Ruthenium red staining: Acidic mucilages, pectins [12,13], and nucleic acids are stained magenta or crimson using this approach (Figure 1a). 1. For 5 minutes, apply 0.1 percent ruthenium red to portions. 2. Remove excess stain by washing parts twice in distilled water. 3. Use glyceringelatin to adhere the parts between the slide and the coverslip.

Alcian Blue Staining:
This test produces results similar to ruthenium red, staining acidic mucilages, pectins ,and nucleic acids light blue 1. For 30 minutes, stain portions with 1% Alcian Blue. 2. Remove excess stain by rinsing areas twice with distilled water. 3. Use glyceringelatin to mount the slide.

Tannic Acid and Ferric Chloride:
This approach is based on the reaction of tannic acid with mucilages and pectins, which are then revealed by ferric chloride, resulting in a grey to black hue ( Figure 1c).

Triple Staining for Starch Detection:
This triple staining was created to simultaneously evaluate structural tissue components and starch grains .Starch grains turn black, acidic compounds (such as nucleic acids and lignin) turn brown, and nonlignified cell walls turn green when safranin, astra blue, and iodine-potassium iodide solution are applied 1. For 1 minute, stain the pieces with 1 percent safranin. 2. Remove excess discoloration by rinsing three times in 50 percent ethanol for a few seconds each time.
3. For 1 minute, stain with 1% astra blue. 4. Remove excess stain by washing three times in distilled water for a few seconds each time. 5. Wait 10 minutes before applying the iodine-potassium iodide solution. 6. Quickly dip pieces in distilled water. 7. Fill the slide with the least amount of water possible.

Carbohydrates PAS Reaction (Periodic Acid: Schiff's reagent):
The Schiff's reagent reveals carbonyl groups formed when periodic acid reacts with carbohydrates, generating carbonyl groups. Carbohydrates have a magenta colour 1. For 30 minutes, apply 1 percent sodium tetraborate (freshly produced). 2. Submerge parts in 1% periodic acid for 10 minutes. 3. Rinse with distilled water for a few seconds. 4. In the dark, apply Schiff's reagent for 15 minutes. 5. Soak the parts for 10 minutes in sodium metabisulfite. 6. Rinse for 10 minutes with tap water. 7. Use glyceringelatin to mount the slides. 8. Repeat the test with the exception of step 2 as a control (periodic acid).

Aniline Blue Staining:
This staining identifies callose, which emits a green fluorescence when exposed to UV light 1. For 10 minutes, apply 0.05 percent aniline blue. 2. Rinse with distilled water for a few seconds. 3. Place the slide in the same staining buffer as before.

Calcofluor White Staining:
This assay is used to detect cellulose in cell walls, which emits a light blue fluorescence when exposed to UV light 1. Dip pieces for 10 minutes in 0.01 percent calcofluor white. 2. Rinse with distilled water for a few seconds. 3. Submerge the mount in distilled water.

Proteins Aniline Blue Black Staining:
This stain identifies blue proteins, whether structural or active in primary or secondary metabolism 1. For 1 minute, dip sections in 1 percent aniline blue black. 2. Remove excess discoloration by washing twice in 0.5 percent acetic acid. 3. Rinse with distilled water for a few seconds. 4. Dehydrate portions by swiftly running them through 90 percent ethanol, 100 percent ethanol, a 1:1 mixture of 100 percent ethanol and xylene, and ultimately pure xylene. 5. Use synthetic resin to mount slides. 6. Control: Before staining, soak sections in a solution of acetic anhydride and pyridine (4:6, v/v) for 6 hours.

Coomassie Blue Staining:
This approach dyes proteins blue and yields a result similar to aniline blue black.

Lipids Sudan Black Staining :
This is a typical approach for staining lipids, which results in a dark blue to black coloration 1. Stain for 20 minutes with Sudan black B. 2. . Rinse with 70% ethanol for a few seconds. 3. Use distilled water to clean. 4. Apply glyceringelatin on the surface. 3 Control: Depending on the content of the secretion, sections should be maintained in the extraction solution for 6 hours or more (determined empirically). Following this time, the portions should be transferred to distilled water and cleaned for 4 hours (4 1 hour). The staining will next proceed as specified.