实验动物技术

Dye Filling to Stain Amphid and Phasmid Neurons by Michael Koelle from Beth Sawin 4/6/94 1. Buy DiO from Molecular Probes catalog # D-275. DiO fluorescesces green (use FITC filters). If you want red f ...

Fixation and Staining ParametersAntigen Fixation Fixation time AntibodyDilutionRefmicrotubules 100% MeOH 5 min @ RT DM1a1:500actin 4% Formaldehydundefined 8 min @ RT C41:5001POD-1 Formaldehyde or MeOundefined 8 min ...

EM Fixation of EmbryosAdapted following advice from Andy Fire and Jim Priess1. Mount embryos on slides as if for DIC except agar has fixative. Agarose Fixative 3ml 2% Agarose : 1.5% LGT Agarose + .05% ...

Bloomington No-Plug Food Prep Method Standard medium in use at Bloomington Hard agar medium in use at Bloomington Cornmeal Sucrose Dextrose Yeast and 2-Acid Medium Cornmeal-molasses-yeast medium Co ...

Gonad Dissections- From R. Francis -1) Pick adults and/or L4s to an unseeded NGM plate. Alternatively wash worms off a seeded plate with 1.5 ml of PBS and allow to gravity settle for a few minutes in ...

Gonad Fixation - From R. Francis -A. Methanol Methanol fixation is quick easy and gives satisfactory nuclear morphology after DAPI staining. However methanol-fixed gonads break easily and are more dif ...

In situ hybridizationIn situ hybridizations were based on a modified method of Seydoux and Fire (1994 Development 120 2823-2834; 1995 Methods in Cell Biology) (H. Tabara and Y. Kohara personal commun ...

FREEZE-CRACK b-GALACTOSIDASE STAINING PROTOCOLFIRE LAB1. Prepare humidity chamber: line large square plate with humid paper towels and place 4 5ml plastic pipets across chamber to support slides.Place ...

Preparation of single-stranded probes from cloned cDNA(from Patel and Goodman 1992)a) 2-5 µg of plasmid DNA containing the cDNA insert is linearized using an appropriate restriction enzyme. For antise ...

MMS integration (both methods modified by Herman Lab) NOTES: Remember that MMS is a potent mutagen -- wear gloves and be very careful. Integration is best done using an array that is transmitted at ve ...

Nematode dye filling(Ed Hedgecock ) Thumb plate Transfer worms into a known amount of M9 in a labeled depression. Add DiO(2 mg/ml in DMF) at 10 l/ml of M9. Let sit for 2 hours. Transfer worms with a P ...

Whole-mount in situ hybridization for the detection of RNA in C. elegans embryosGeraldine Seydoux and Andrew Fire Adapted from Seydoux G. and Fire A. (1995). Whole-mount in situ hybridization for the ...

"CONVENTIONAL" TWO STEP FIXATION 1995 1. Wash animals in M9 and anaesthetized in 8% alcohol in M9 5’ 2. 2.5% glutaraldehyde 1% paraformaldehyde in 0.1M sucrose 0.05M cacodylate on ice 2 hours 3. 3 ri ...

Phalloidin Staining worms by Michael Koelle modified from Beth Bucher and Andrew Chisholm 4/6/94 Phalloidin binds to filamentous actin. This stain allows visualization of mainly of muscles but some ot ...

Staining C. elegans for ß-galactosidase activity using X-gal by Michael Koelle adapted from Laird Bloom Jeff Way & Michael Basson 4/6/94 1. Grow up a small plate of worms. Some protocols call for usin ...

Embryo BuffersEgg Salts118 mM NaCl 48 mM KCl 2 mM MgCl2 2 mM CaCl2 10 mM HEPES (pH 7.4) ...

Cuticle preparationsThis procedure was adapted from one described over the phone (thanks P.G.). We're not sure if it appears elsewhere in published form.-Collect embryos on apple juice-agar plates for ...

GENERAL MAINTENANCE INSTRUCTIONS FOR IMAGINAL DISC CELL LINES This is our lab protocol for growing imaginal disc cells (clone-8) The Milner Lab website has additional protocols for clone-8 cells. Cl ...

Medium Shields and Sang Medium 3 (SS3) modified for low serum. Supplemented before use with 2% heat inactivated FCS 2.5% FE2 12.5IU/100ml insulin and filter-sterilised through 0.22µm filter. Supplemen ...

SINGLE-FLY DNA PREPS FOR PCRGloor et al. 1993 Genetics 135:81-95We have developed a simple method for the rapid and reproducible isolation of DNA from single flies for amplification by the polymerase ...