Experimental experiments on the coating preparation of raw foaming inclusions in the African clawed toad (Clavicipitus africanus)

Summary

This experiment is based on the "Guide to Cellular Experiments", translated by Huang Peitang et al.

Operation method

Preparation of African clawed toad smears

Materials and Instruments

Female African clawed toad
OR2 Culture Solution "5:1" Separation Solution Dispersant
Petri dish

Move

1. A piece of ovary from an anaesthetized female Xenopus laevis was placed in a Petri dish (60 mm x 15 mm) containing OR2 culture solution at a temperature of 18-22 °C.

OR2 culture solution:

82.5 mmol/L NaCl

2.5 mmol/L KCI

1.0 mmol/L _CaCl2

1.0 mmol/L MgCl₂

1.0 mmol/L _Na2HPO4

5.0 mmol/L HEPES

2. Separate individual oocytes or small piles of oocytes with jeweler's tweezers and transfer to a fresh OR2 dish. Leave at 18-22°C (not at 4°C) for 18-24 hours. At the end of this time, almost all oocytes, up to the largest oocytes, have transcriptionally active chromosomes containing large numbers of light brush chromosome rings. The use of gonadotropins as recommended previously for the pretreatment of female African clawed toads is not necessary.

3. Select a 1.0-1.1 mm diameter oocyte and place it in a small Petri dish (35 mm x 15 mm) containing a "5:1" isolate. Using two jeweler's tweezers, make a large tear in the animal hemisphere (black hemisphere), locate the hair follicle in the protruding cytoplasm and dislodge it.

"5:1" isolation solution, pH 7.0

83.0 mmol/L NaCl

17.0 mmol/L KCl

6.5 mmol/L _Na2HPO4

3.5 mmol/L _KH2PO4

1.0 mmol/L MgCl₂

1.0 mmol/L DTT

4. Transfer the complete raw foam to a petri dish containing dispersant within 20 seconds.

Dispersant:

20.7 mmol/L NaCl

4.3 mmol/L KCl

1.6 mmol/L _Na2HPO4

0.9 mmol/L _KH2PO4

1.0 mmol/L MgCl₂

0.01 mmol/L _CaCl2

1.0 mmol/L DTT

0.1% paraformaldehyde

5. In a quick procedure, remove the nuclear membrane with jeweler's tweezers and transfer the still-intact nuclear inclusions to a dispersant-filled microscope slide coating chamber, add a coverslip, and seal with petroleum jelly. The cell membrane inclusions will disperse and settle to the bottom of the chamber within a few minutes.

6. The slide is centrifuged at approximately 5000 g for 30 minutes at 5-10°C.

7. After centrifugation, the slide is placed vertically in a stained Petri dish containing PBS + 2% formaldehyde. Push the coverslip flat and leave it there for 1-2 hours without moving it.

8. At this point, if the nuclear inclusions are firmly affixed to the slide, remove the coating chambers with a razor blade.

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