Slides, mounting

Mount and coverslip Mount slides using clean cover slips and dry overnight. Slides can be cleaned the following day using xylene or chloroform. Remember, it is important to remove all bubbles from under the cover slip. Be conservative with the amount of Permount used in mounting, as using too much can obstruct the view of the sections under a microscope. 

As part of a periodic maintenance schedule, the volume of fluid dispensed by the pipeter should be quantitatively assessed to detect any restricted flow that might occur owing to blockage of the pipeter. If double-mounted slides are used, with... 

Mount slides directly with anti-fade ProLong Gold containing DAPI. Store slides dark and at 4°C. Evaluate staining result after 24 h. 

Add 60 xL of DAB substrate solution and leave for 5 min at room temperature. Rinse the slides in tap water to stop the reaction (j Note 10). Counterstain for 5 min in 10% Giemsa, rinse in buffer, and air-dry (se Note 11). Permanent mounting without subsequent foding of the chromosome and/or hybridization signal is best achieved by mounting slides in Xam. 

Mounting Slides are made permanent by covering the specimen with a noninterfering mounting medium and a number-one coverslip. 

Dehydrate and mount slides in Antifade with PI and DAPI (see Note 8). 

Mounted slides were observed with the Nikon Eclipse microscope under the bright light or under the epi-fluorescence using Rhodamine/TRITC filter (Fig. 1). 

Prepare a wet mount slide for DIC microscopy. Melt 2% agarose in H O by microwaving, and place a 1-cm diameter drop in the center of a microscope slide. 

To mount slides for microscopy, remove as much liquid as possible without allowing the sample to dry out. Pipet a small drop of mounting medium onto a 22 X 22-mm cover slip (or slide if the sample is on a cover slip). Touch the specimen to the drop of mounting medium, invert the slide, and seal with nail polish. 

Histolene used for the initial dewaxing of sections can be kept for use in mounting slides in DePex. 

Mount slides in Vectashield H-1000 (Vector Laboratories) or similar antifading mountants. Seal the coverslip edges with either rubber cement or nail polish and store in the dark at 4°C. 

Mount slides in 80% glycerol in PBS or dehydrate through a graded ethanol series (once in 30%, 50%, 70%, 90%, and twice in absolute 10 minutes each) and mount in DPX (Fluka). 

Custom-designed mounting slides. Use a razor blade to cut a square opening in a piece of colored tape on a coverslip (24 X 40-mm, 1.5). This opening acts as a well, and the sides of the tape provide support for an overlying permeable membrane. Coat the bottom of the well with heptane glue that is allowed to desiccate before embryos are applied to its surface. 

Place the embryos in the center of the well of a custom-designed mounting slide. Cover with halocarbon oil. Place a permeable membrane over the embryos and the sides of the tape. 

Wash sections with PBST 6 times for 10 min each and incubate with the secondary antibodies 1 250 in PBST for 90 min. Protect slides from light from this step on. Wash sections with PBST three times for 10 min each and then incubate with DAPI working solution for 15 min. Finally, wash sections with PBST six times for 10 min each and mount slides with Fluoromount . Once mounted remember to seal each slide with nail polish. 

Nonspecific binding of antibodies can be inhibited with 5 % BSA in PBS. Ready-to-use blocking solutions may be used for this purpose as well. In the case of manual processing of preparations, to minimize volume of the reagents applied to the horizontally mounted slides, a special pen is necessary to outline the section on the slide with a hydrophobic circle. For long-term incubation of... 

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