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Protocol for Nile Red Staining - Fly
Author: Jennifer Beck, updated date: , view: 36, Q&A: 0
Tags: Microscopy, Fly and Drosophila

Procedure

  1. Stock Solution:

    • PBS

    • Fix solution (freshly prepared): 4% paraformaldehyde (PFA) in PBS

    • PBX: PBS, 0.1% Triton X-100

    • Nile Red (500 ul/ml) dissolved in acetone 

  2. Method:

    • Dissect flies in PBS 

    • Fix with 250-300 ul of 4% PFA in PBS for 30 min at room temperature (RT) 

      • 100 mg PFA + 1 ul NaOH + 500 ul H2O --> 20% stock (2-3 min @ 65°C to dissolve) 

      • 50 ul of 20% stock to 200 ul PBS 

    • Wash with 300 ul of PBS for 10 min at RT x3 times. 

    • Incubate with 250 ul of 1 ug/ml Nile Red (0.5 ul NR/sample, 250 ul) in PBX for 2 hrs at RT or overnight at 4°C 

    • Wash with 300 ul of PBX for 5 min at RT x2 times 

    • Incubate with 300 ul of DAPI in PBX (1:4,000 dilution) for 20 min at RT 

    • Wash with 300 ul of PBX for 10 min at RT x3 times 

    • Wash with 300 ul of PBS for 5 min at RT x2 times 

    • Mount on the slide glass with Mowiol medium [M] 

      • align all tissues facing up

      • 1x drop of medium (~35ul]

      • add cover slip

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