Hemolymph-brain barrier permeability

Adapted from Pinsonneault et al., 2011 “Novel Models for Studying the Blood-Brain and Blood-Eye Barriers in Drosophila"

1. Prepare injected solution: dilute AF647-10kd dextran to 10mg/ml in PBS
2. Prepare tapered injection needles by pulling thin-walled glass capillaries using a needle puller (pulled with Narishige PC-10) 
3. Break tip of needle with scissors or against a glass slide 
4. Fill needles with prepared solution by leaving the needle tip in solution. This should fill by capillary action 
5. Attach needle tip to appropriate tubing to adapter to 3-way stopcock to Luorlock 10ml syringe 
6. Anesthetize flies on fly pad with CO2 
7. Visualize needle and flies under stereoscope 
8. Slide needle into the center of the protuberance of the left prescutum region of the fly's thorax 
9. Apply gentle positive pressure to the needle. Fly should turn blue. Injections should be approximately 30-50 nL volume – this can be measured using a plate reader on a subset of injected flies 
10. Allow injected flies to recover on standard food vials overnight
11. Anesthetize flies on fly pad with CO2 
12. Remove heads and proboscis and immediately fix in 4% PFA in PBST (PBS + 0.1% Triton)  for 10–15 min 
13. Dissect out brains in PBST and remove trachea under stereoscope 
14. Wash brains in PBST for 30 mins 
15. Transfer brains to 70% glycerol in PBST solution
16. Mount brains in VectaShield (Vector Laboratories, H-1000), carefully lower coverslip with forceps 
17. Image slides with confocal Leica SP5

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