Some Tips for Salivary Gland Dissection and Preparing Nice Slides

• Reagend 4 should be at least 50 ml although 60 ml is better to not let the coverslip stick to the slide while swirling it.
• Reagend 4 should not be less than 50 ml, otherwise there will be air bubbles at the edges of the coverslip which will result in crystalization of the sample because of vaporization of acetic acid.
• Reagend 2 and 3 should be fresh meaning at the end of each hour they should be reprepared. Each 30 ml of these reagends should also be changed after 10 minute.
• Reagend 3 and 4 should be kept their caps closed because not to let acetic acid to evaporate.
• Before swirling the coverslip on the slide, you should press gently on where the salivary glands are. This is to make nucleus come out of the cells and stick to the slide.
• Swirling makes the chromosomes spreaded on the glass slide. Therefore to have nicely spreaded samples you should not afraid to push while swirling. After some time of experience the optimum force will be found.
• Salivary glands should not be sepereated from each other and they should be placed at the center of the slide in the reagend 4. This is for having a big number of chromosomes in a single slide.
• The edges of the coverslips should be marked to know where is the sample on the slide. This will help during primary and secondary antibody staining. 50 ml of primary and secondary antibody solutions will be enough for one slide.
• The slide and the coverslip should be cleaned just before dissection and must be dust free.
• The last pushing step after turning the slide upside down is for to make chromosomes stick to the slide.
• After placing the slide in nitrogen coverslip should be removed immediately and fastly (in one shot) with scalpel.
• Slides might be kept in PBST at 4°C overnight.
• Reagends 2 and 3 can be used for max 2-3 hours. Always prepare fresh is dissecting for longer time.
• Reagends 2 and 3 could be prepared in large volumes without adding paraformaldehyde. Just before using small volumes can be prepared by adding proper volume of 16% paraformaldehyde.
• The best is to keep animals on 18°C, the salivary glands will be better developed.
• Starting from L2 stage larvae shoul be fed with yeast.
• The tubes should not be very crawded to let larvae develop nicely.
• Salivary glands that are balloon like and transparent shouldn’t be processed. They will not give nicely spreaded chromosomes.
• To have nice salivary glands and nice slides with well spreaded chromosomes, moving larvaes should be selected.