Paraffin Embedding of Drosophila Pupal Tissues
Puncture posterior end of staged pupa with dissecting scissors , fine forcepts , or . 22 gauge needle .
Place into Eppendorf tube with 500 ul of FAAG and store overnight at 4 degrees Celsius .
Remove FAAG in the hood and dispose in " Aldehyde Waste " container .
Dehydrate through ethanol series and clear in Xylenes .
Rotate in nutator at RT .
Solution 1 : 80 % Ethanol 500 ul Dehydrate through ethanol series continued .
Solution 2 : 85 % Ethanol 500 ul Dehydrate through ethanol series continued .
Solution 3 : 90 % Ethanol 500 ul Dehydrate through ethanol series continued .
Solution 4 : 100 % Ethanol 500 ul .
Repeat Step 5 Dehydrate through ethanol series continued .
Solution 4 : 100 % Ethanol 500 ul .
Repeat Step 5 Dehydrate through ethanol series continued .
Solution 4 : 100 % Ethanol 500 ul .
Repeat Step 5 Dehydrate through ethanol series continued .
Solution 4 : 100 % Ethanol 500 ul .
Replace 100 % Ethanol with 500ul Xylenes for thirty minutes .
Dispose of Xylenes in " Aldehyde Waste " container .
Repeat Step 9 : Remove Xylenes and replace with new 500ul Xylenes for thirty minutes .
Dispose of Xylenes in " Aldehyde Waste " container .
Repeat Step 9 : Remove Xylenes and replace with new 500ul Xylenes for thirty minutes .
Dispose of Xylenes in " Aldehyde Waste " container .
Repeat Step 9 : Remove Xylenes and replace with new 500ul Xylenes for thirty minutes .
Dispose of Xylenes in " Aldehyde Waste " container Paraffin Infiltration .
Transfer pupae to scintillation vial with 2 ml of Xylenes using designated paintbrush .
Mark the level of Xylenes on tube .
Add ~ 20 paraffin chips ( paraplast xtra ) and melt in 42 degree Celsius incubator .
( Volume should double ) Leave overnight .
In 500 ml beaker , add melted paraffin and incubate at 56 degrees celsius for use tomorrow .
Remove Xylenes / Wax and place onto wax waste tray .
Add a small amount of melted paraffin at 56 degrees Celsius to dilute residual Xylene , remove and place onto wax waste tray .
Add melted 56 degree Celsius paraffin incubate for 1 - 2 hours .
Remove paraffin and replace with fresh melted 56 degree Celsius paraffin incubate for 1 - 2 hours .
Remove paraffin and replace with fresh melted 56 degree Celsius paraffin and fill to the top of the glass vial .
Incubate overnight .
Make mold by taping the ends and sides of cut index cards and tape ends to prevent leaks .
Pre - heat hot block to 56 degrees Celsius and cover completely with tin foil .
When hot block reaches temp , swirl pupae in the vial and quickly pour wax and pupae into mold on top of hot block .
Orient the samples quickly before the wax hardens .
Fill the remainder of the boat with melted paraffin .
Let sit for 15 minutes to begin to solidify .
Carefully remove boat from hot block and coll overnight at room temp .
Cut wax into individual pyramid shaped blocks for sectioning using a razor blade .
Mount blocks onto chuck by melting paraffin with heated flat spatula .
Cection blocks on microtome at 7um and float onto slides with water that are pre - warmed on slide warmer at 42 degrees Celsius .
Allow the sections to expand on slide warmer for 2 - 5 minutes .
Remove water by wicking with a kimwipe .
Bake slides on 42 degree Celsius slide warmer overnight .
Hydrate samples by placing in Xylenes ( container 1 ) for 3 minutes .
Hydrate samples by placing in Xylenes ( container 2 ) for 3 minutes .
Hydrate samples by placing in 100 % Ethanol ( container 1 ) for 3 minutes .
Hydrate samples by placing in 100 % Ethanol ( container 2 ) for 3 minutes .
Hydrate samples by placing in 95 % Ethanol for 3 minutes .
Hydrate samples by placing in 70 % Ethanol for 3 minutes .
Rinse under running tap water for 5 mins .
Stain Samples Submerge slides in Weigert's Hematoxylin for 5 mins .
Rinse under running tap water for 5 mins .
Submerge in Pollak Trichrome for 7 mins .
Rinse in distilled water for approx 10 seconds to remove the bulk of the stain .
Submerge in Acidified distilled water for 10 sec .
Dehydration : Dip slides into 70 % Ethanol 3 times .
Dip slides into 95 % Ethanol ( container 1 ) 3 times .
Submerge slides into 95 % Ethanol ( container 2 ) for 3 mins .
Submerge slides into 100 % Ethanol ( container 1 ) for 3 mins .
Submerge slides into 100 % Ethanol ( container 2 ) for 3 mins .
Submerge slides into Xylenes ( container 1 ) for 3 mins .
Submerge slides into Xylenes ( container 2 ) for 3 mins .
Remove slides from Xylenes and drain for 2 mins on a paper towel Use 78ul of Permount ( for large coverslip ) and apply coverslip .
Let slides harden overnight .
