Wednesday, May 26, 2010

Salivary Gland Dissection






Reagend 1:  28 mikrol of 10% NP-40
                   50 mikrol 10X PBS
                   422 mikrol ddH2O        
Reagend 2:  40 mikrol 10X PBS
                   100 mikrol 16% paraformaldehyde
                   80 mikrol 10% NP-40
                  180 mikrol ddH2O
Reagend 3: 180 mikrol of 100% acetic acid
                   120 mikrol ddH2O
                   100 mikrol 16% paraformaldehyde
Reagend 4:  450 mikrol of 100% acetic acid
                   550 mikrol ddH2O
Dissection:
Dissect salivary glands from wondering L3 larvae in 30 mikrol of Reagend 1.
Remove fat body from the glands.
Transfer the glands to mikrol ml Reagend 2, incubate for 40 seconds.
Transfer the glands to mikrol ml Reagend 3, incubate for 40 seconds.
Transfer the glands to 50 mikrol Reagend 4, that is placed in a slide, incubate for 1 min.
Cover with cover slip, gently push with a finger and move in a circle.
Turn the slide upside down, place in a layer of paper tissue, push vigorously.
Place the slide in liquid nitrogen and remove cover slip with scalpel.
Incubate slide in PBST (PBS + 0.1% Tween20)




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