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The cells were washed twice in PBS and then mounted by inverting onto mounting medium on glass slides.
Following incubation, the cover slips were washed five times in PBS and mounted in Vectashield mounting medium on glass slides.
After 5 days, the cover slips were fixed for 7 min in 4% paraformaldehyde, washed 3 times with PBS, and then incubated with 0.1% DAPI for 5 min. Following incubation, the cover slips were washed five times in PBS and mounted in Vectashield mounting medium on glass slides.
After PBS washing, coverslips with mounting medium on tissue slides were prepared for investigation by Nikon E800 fluorescence microscopy.
Fat bodies were transferred to Vectashield (Vector Laboratories, Burlingame, CA, USA) mounting medium on glass slides, covered and immediately imaged on a Zeiss LSM510 confocal microscope.
Cells were fixed with 90% acetone (10 min) and then were treated with human CD31 (1/100, Invitrogen, 45 min) and Alexa 488 + Hoechst 33258 (30 min) and covered with mounting medium on slides.
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Extracted ML and control tonsillar nuclei were stained with DAPI and mounted with Vectashield [Vector Laboratories, Inc. Burlingame, CA, USA] mounting medium for fluorescence microscopy on SuperFrost® slides.
After washing, the tissue sections were mounted with VectaMount mounting medium (Vector Laboratories, Burlington, ON, Canada) and examined with light microscopy.
We removed wings from 3- to 8-d-old adult flies (here we examine females only) using forceps (Dumont #5 Forceps, Fine Science Tools; www.finescience.com) and transferred five to six wings to a drop of Euparal mounting medium (Bioquip.com) on a clean cover slip (Fisherbrand Superslip Cover Slips; Fisher Scientific).
After extensive washing with PBS, the coverslips were mounted on slides with Dako fluorescent mounting medium (Dako Canada, Mississauga, ON, Canada).
Tissue sections were mounted on slides and allowed to dry for at least two hours, dehydrated through an ascending alcohol series, cleared in Histoclear (Diamed, Mississauga, ON, Canada), and coverslipped with Entellan mounting medium (BDH Chemicals, Toronto, ON, Canada).
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