Orcein

Orcein dyes bind to negatively charged groups of chromatin or through hydrophobic interactions under acidic conditions, staining chromosomes. Orcein is mainly used to stain polytene chromosomes in the salivary glands of third-instar larvae of Drosophila. It can also be used to observe chromosomes in tissues such as the midgut, hindgut, and fat body^[1].
Orcein can be used to stain tissue samples. In histology and cell biology, it is mainly used to stain elastic fibers, collagen, nucleic acids, and cellular components^[2].

Guide (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs)^[1].
Aceto-orcein staining:
When staining with Orcein, adding acetic acid to fix the chromosomes can stretch the chromosomes in the interband region when pressing the slides, improving the band resolution; the addition of lactic acid can soften the glands and promote chromosome spreading.
1. Materials
1.1 Culture medium: Texas banana agar, yeast-glucose-agar, Carolina instant Drosophila feed.
1.2 Buffer/fixative: Drosophila Ringers solution, PBS, 0.8% NaCl, 45% acetic acid.
1.3 Staining solution: lactic acid-acetic acid-orcein staining solution (prepared according to Lim's method: 1 g natural orcein dissolved in 50 mL lactic acid, filtered; 1 g natural orcein dissolved in 50 mL glacial acetic acid, heated and filtered; the three were mixed at a ratio of 1:1:1).
1.4 Consumables: No. 5 tweezers, siliconized coverslips, coated slides, Probe-On-Plus slides, Permount mounting medium.
2. Operation steps
2.1 Larva culture and selection:
Use the above culture medium to culture third-instar larvae. Cultivate at 18°C to obtain larger chromosomes. Select larvae that have not pupated and are full-bodied, and rinse the culture medium on the body surface with PBS or Ringers solution.
2.2 Dissection and gland separation:
Place the larvae on a slide containing 45% acetic acid. Under a dissecting microscope, use a pair of forceps to clamp the head and tail respectively, and pull the head off quickly to expose the salivary gland (a transparent long cyst-like structure with attached adipose tissue).
Strip the gland, remove the anterior duct and excess fat, and fix in 45% acetic acid for 2-5 minutes.
2.3 Staining and pressing:
Transfer the gland to lactic acid-acetic acid-orcein staining solution and stain for 5 minutes (avoid evaporation of the staining solution).
Transfer to a slide containing 1:2:3 fixative, cover with a coverslip, tap the coverslip with a dissecting needle to spread the chromosomes, and then press lightly with absorbent paper for blotting (to prevent the coverslip from shifting).
2.4 Seal and observe:
Temporary seal: seal the edges of the coverslip with nail polish; permanent seal: peel off the siliconized coverslip after freezing with dry ice-ethanol, dehydrate with 95% and 100% ethanol in turn, and seal with Permount.
Microscope observation: identify chromosome arms (such as X, 2L, 2R, 3L, 3R) by telomere morphology and banding characteristics (such as puff, constriction area).
3. Precautions
3.1 45% acetic acid fixation can reduce chromosome puffing, which is better than physiological saline dissection.
3.2 Avoid excessive force when pressing the slice to cause chromosome breakage. Light spiral or zigzag pressure can promote uniform spreading.
3.3 Different fruit fly strains and culture conditions may lead to differences in banding patterns, which need to be combined with standard pattern analysis.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.