Troubleshooting guide

1. Individual sections fall apart during sectioning with no ribbons formed. Overfixation of tissues could have occurred. Try a shorter fixation time and determine an optimal fixation time.

2. Sections crumble and are damaged during stretching. Underfixation of tissues has occurred. Use a longer fixation time and determine the optimal time empirically.

3. Sections have holes. This is due to incomplete tissue dehydration and improper impregnation with wax. Perform complete dehydration of fixed tissues followed by thorough impregnation with melted paraffin wax.

4. Holes occur in frozen sections. Ice crystals have been formed in the tissues during freezing or in the cryostat. Make sure to freeze the tissue correctly. Do not touch the frozen sections with fingers or relatively warm tools while mounting the specimens in the microtome or brushing the sections from the cutting knife.

5. Sections are not of uniform thickness or have lines throughout. The cutting surface of the specimen block is not parallel to the edge of the knife or the knife is dull. Take great care to use a sharpened knife while mounting the specimens in the microtome.

6. Morphology of tissue is poorly preserved. Tissues are stretched or shrunken. Make sure not to press or stretch the tissue during dissection and embedding.

7. The specific activity of a labeled probe is <105 cpm/mg DNA or RNA.

[35S]dCTP for DNA or [35S]UTP for RNA has passed its half-life time. The activity of DNA or RNA polymerase is low or may be inhibited. Try to use fresh isotope and ensure that the labeling reaction is set up under the optimal conditions, including high-quality DNA

8. A low signal is evident. Tissue is underdigested with protease. The specific activity of the enzyme is low. Determine the optimal digestion time of the specimens. Make sure that the probe is good.

9. A high background occurs. Tissue is overfixed. Protease digestion of the tissues is not sufficient. Set up a series of fixation times. Try to use an optimal digestion.

10. A good signal is evident but poor morphology occurs. Tissue overdigestion occurs. Counterstaining the tissue is not sufficient. Tissue should be properly digested with an appropriate protease. Counterstain the sections slightly longer.

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