Jul 25, 2015
Paraffin Embedding of Drosophila Pupal Tissues
- Sonia M. Hall1
- 1University of Massachusetts Medical School
Protocol Citation: Sonia M. Hall 2015. Paraffin Embedding of Drosophila Pupal Tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.dbb2im
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: July 08, 2015
Last Modified: March 06, 2018
Protocol Integer ID: 1091
Keywords: drosophila pupal tissues drosophila tissue processing, paraffin embedding
Abstract
Drosophila Tissue Processing for Paraffin Embedding
modified by the Baehrecke Lab from published protocols by Ristifo and White, 1991 (Pubmed ID 1936557) and Muro et al., 2006 (Pubmed ID 16887831)
Troubleshooting
Before start
Collect white pre-pupae, raised at 25 degrees Celsius, every forty minutes until desired number is obtained. Age for 24 hrs at 25 degrees Celsius and then begin fixation based on the time collected for each individual.
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
500 µL
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
500 µL
00:15:00
Dehydrate through ethanol series continued
Solution 2: 85% Ethanol 500 ul
500 µL
00:20:00
Dehydrate through ethanol series continued
Solution 3: 90% Ethanol 500 ul
500 µL
00:25:00
Dehydrate through ethanol series continued
Solution 4: 100% Ethanol 500 ul
500 µL
00:30:00
Repeat Step 5
Dehydrate through ethanol series continued
Solution 4: 100% Ethanol 500 ul
00:30:00
Repeat Step 5
Dehydrate through ethanol series continued
Solution 4: 100% Ethanol 500 ul
00:30:00
Repeat Step 5
Dehydrate through ethanol series continued
Solution 4: 100% Ethanol 500 ul
00:30:00
Replace 100% Ethanol with 500ul Xylenes for thirty minutes.
Dispose of Xylenes in "Aldehyde Waste" container
500 µL
00:30:00
Repeat Step 9: Remove Xylenes and replace with new500ul Xylenes for thirty minutes.
Dispose of Xylenes in "Aldehyde Waste" container
500 µL
00:30:00
Repeat Step 9: Remove Xylenes and replace with new500ul Xylenes for thirty minutes.
Dispose of Xylenes in "Aldehyde Waste" container
500 µL
00:30:00
Repeat Step 9: Remove Xylenes and replace with new500ul Xylenes for thirty minutes.
Dispose of Xylenes in "Aldehyde Waste" container
500 µL
00:30:00
Paraffin Infiltration
Transfer pupae to scintillation vial with 2 ml of Xylenes using designated paintbrush.
Mark the level of Xylenes on tube.
2 µL
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.
01:30:00
Remove paraffin and replace with fresh melted 56 degree Celsius paraffin incubate for 1-2 hours.
01:30:00
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.
00:15:00
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
00:03:00
Hydrate samples by placing in Xylenes (container 2) for 3 minutes
00:03:00
Hydrate samples by placing in 100% Ethanol (container 1) for 3 minutes
00:03:00
Hydrate samples by placing in 100% Ethanol (container 2) for 3 minutes
00:03:00
Hydrate samples by placing in 95% Ethanol for 3 minutes
00:03:00
Hydrate samples by placing in 70% Ethanol for 3 minutes
00:03:00
Rinse under running tap water for 5 mins.
00:05:00
Stain Samples
Submerge slides in Weigert's Hematoxylin for 5 mins.
00:05:00
Rinse under running tap water for 5 mins.
00:05:00
Submerge in Pollak Trichrome for 7 mins.
00:07:00
Rinse in distilled water for approx 10 seconds to remove the bulk of the stain.
00:00:10
Submerge in Acidified distilled water for 10 sec.
00:00:10
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.
00:03:00
Submerge slides into 100% Ethanol (container 1) for 3 mins.
00:03:00
Submerge slides into 100% Ethanol (container 2) for 3 mins.
00:03:00
Submerge slides into Xylenes (container 1) for 3 mins.
00:03:00
Submerge slides into Xylenes (container 2) for 3 mins.
00:03:00
Remove slides from Xylenes and drain for 2 mins on a paper towel
00:02:00
Use 78ul of Permount (for large coverslip) and apply coverslip. Let slides harden overnight.
78 µL