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==Solovei Protocol== [[Media:FISH_TissueProtocol.pdf|Original Protocol]] ===Tissue Fixation=== #Fix tissue pieces of an appropriate size in freshly prepared 4% formaldehyde/PBS for 4–24 h #Wash the tissue in PBS, 3 × 30 min. Tissue can be stored in PBS for few days at +4°C #Incubate the tissue in a sucrose series with increasing concentrations: 10% for 1 h, 20% for 1 h, and 30% for 1–24 h. ##The incubation time also depends on the size of the tissue piece. ##Tissue can be stored in 30% sucrose at +4°C up to a few days or can be frozen in this solution for longer storage at −20°C ===Pretreatment=== #Remove slides from freezer, let them thaw and air-dry at RT for 30–60 min ##While the slides are drying, mark the future hybridization area as a rectangular on the back side of the slide using a diamond cutter. These marks will help to localize the hybridized area during the following procedures #Re-hydrate sections in 10 mM sodium citrate buffer for 5 min #Transfer slides into a Coplin jar with prewarmed (80°C) 10 mM sodium citrate buffer in a water bath and incubate there for 5–25 min #Remove the Coplin jar from the water bath and let it cool down at RT #Incubate the slides in 2× SSC for 5 min #Transfer slides into 50% formamide in 2× SSC and incubate for at least 1 h ##sections can be stored in this solution at 4°C for weeks ===Hybridization=== #Prepare glass hybridization chambers as follows (Fig. 2a, b): ##Cut glass strips from a coverslip (e.g., 8 × 8 mm) using a diamond cutter and a ruler; ##Glue glass strips parallel on two opposite borders of an intact coverslip of the same size using nail polish ##Size of the chamber depends on a section size: chamber should fit to the section size as closely as possible in order to save DNA probe ##Let the nail polish completely dry before use #Remove slide from the formamide and wipe the liquid from the back of the slide and from the face side of the slide ##Avoid marked section area #Place prepared hybridization chamber above the section and carefully press it against the microscopic slide #Load the probe under chamber from its open side so that probe fills in the whole space above the section (Fig. 2c, d) #Seal the chamber with rubber cement ##It is important that rubber cement covers the entire chamber periphery or even the whole chamber (see Fig. 2e) #Let the rubber cement dry at RT ##From this step, protect slide from light #Place slide on a hot-block (or water bath) with temperature 45°C for 1–3 h in order to allow infiltration of the section with the probe #Place slide on a hot-block at 80°C for 5 min for the probe and section denaturation of DNA #Hybridize slide for 2 or 3 days at 37°C in a waterbath or temperature controlled oven [[Image:Figure2_Solovei_TissueFISH.png|800px]] ===Post-Hybidization=== #Remove rubber cement together with the chamber using a pair of fine forceps #Wash slides in 2× SSC at 37°C, 3 × 15 min (preferably with shaking) to remove hybridization mixture #Perform stringency washes in 0.1× SSC at 60°C, 2 × 5 min to remove nonspecific hybrids #Equilibrate in 2× SSC for 2 min #Add DAPI, let sit for at least 30 minutes #Seal the coverslip with nail polish; let sit 30 minutes ##Sections become fragile after the FISH; therefore, strong pressure on the coverslip should be avoided ##e.g., pressing the coverslip with the microscope objective when focusing often completely destroys the sample
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