coating beads with antibodies

1. 20 ug monoclonal antibody 2. 100 ul of 0.5% w/v goat-anti-mouse IgG coated beads, diameter = 4.4 um (~1x10^7 beads) (hint: always count the beads) Incubate mixture (no further dilution necessary) at 4 deg in polypropylene tubes, rotate over night or longer. Before use, wash the coated beads 3x in PBS. Beads are purchased from Spherotech (Gt-antiMs-IgG(Fc) Ferromagnetic Particles, catalogue # FMFc-40-5) Neutrophil CD-45 AB's are purchased from PharMingen. For rat anti-CD45, the catalogue # is 22131D, and for human, the catalogue # is 31251A. We started to test our own solid ferromagnetic beads coated with streptavidin (covalently coupled with cross-linked BSA , done by Clemente Associates Inc.). For a procedure on coating those beads with biotinylated antibodies, see the protocol (PDF-format) supplied by Clemente Inc.

coating wells

1. carbonate buffer 2. collagen I (brand-name "Vitrogen" from Cohesion (Palo Alto, CA), or fibronectin 5 ug collagen or fibronectin in 1 ml carbonate buffer (this corresponds approximately to a coating density of 500 ng per cm2 when added to 96-wells) add 100 ul to each 96-well, store at 4 deg over night (The collagen stock solution has a concentration of 3 mg/ml and is usually diluted 1:10 in carbo buffer first; this intermediate "stock" can be stored for several month)

Costaining protocol for alpha-actin and actin

PFA: Paraformaldehye, 2% in PBS, containing 5% cytoskeletal extraction buffer Cytoskeletal extraction buffer: (0.5% TRITON-X, 50mM NACl, 300 mM sucrose, 3 mM MgCl2, 20ug maprotinin, 1 ug/ml leupeptin, 1ug/ml pepstatin, 1mM PMSF, 10 mM Pipes, pH 6.8) Dilution buffer: (0.1% BSA, 0.05% Tween in PBS). Blocking buffer: 10% BSA in PBS, or skim milk powdered 5% in PBS Mounting media: antifade (Molecular Probes) Fixation:PBS and PFA at 37 deg C for SMC, (but preferably ice cold PFA for other cells) optional: wash 1x with PBS, aspirate completely, Add PFA for 15 min can be refrigerated 4 C in PBS for several days if necessary Staining alpha-actin anti-mouse: Wash 2X PBS Permeabelizing: Add TRITON-X 0.3% (even though it's in cyto Buffer) in PBS 5 min. Rinse 2X PBS Block with blocking buffer, 2 hr, room temp. Aspirate Add primary antibody (diluted in dilution buffer at 1:400) 1 hour, (or overnight, does not seem to make a difference), covered (aluminum foil) and humidified. Humidified by placing wells in a dish over a wet towel in a tray, room temp wash 4X PBS, (let washes sit successively longer times, 15'-30' total). apply secondary AB (Texas Red, anti mouse) (diluted in dilution buffer 1:200). Incubate 30' to 2 hr. room temp, covered and humidified. Rinse 3x PBS, (let washes sit successively longer times, 15' total). Coverslip and mount or add further antibody Co-Staining FITC phalloidin (green): (note alexa 488 from Molecular Probes is better, lasts longer and has same wavelength) Aspirate the PBS from last line above Add FITC phalloidin (dilution 1:40 in dilution buffer), or Alexa 488 (dilution 1:100 equals 5 ul methionic stock (5 U) in 200 ul dilution buffer, that's enough for a 22 mm coverslip). For 96 wells, 30ul per well is enough) incubate for 30 min. (4 °C, or room temp.) Wash 4x PBS Coverslip and mount Single staining FITC phalloidin (green): (note alexa 488 is better, lasts longer and same wavelength) Wash 4X PBS Add TRITON-X 0.3% in PBS 5 min. Rinse 2X PBS Not-req'd: [Block with blocking buffer, 1 hour, room temp.] Aspirate Add FITC phalloidin (in dilution buffer 1:40) Incubate for 30 min. covered and humidified (4 °C, or room temp.) Wash 4x PBS IF want nuclear staining, add propitium Iodiode (PI 1ug/ml) 5 min. (note stains red). and rinse 4 X PBS Coverslip and mount Label coverslips for conditions Add 3-4 separate drops (each condition) of mounting media (4 ul) on rectangular coverslips place in top corners and middle of bottom. Extract 5.4 mm dia coverslips from 96 wells with tweezers and sharp pointed spatula. Touch the edge of the 5.4 mm dia coverslip onto kim-wipe tissue to dry by capillary action Invert 5.4 dia coverslips (cell side down) onto drops. let set covered over night at room temperature

general lab-protocols (The WWW Virtual Library of Cell Biology)

iProtocol - A web-based database for protocols used in bioscience based in the Massachusettus Institute of Technology.

Molecular Biology Protocols (by Bart Frank)

I have started to compile a list of all items I oder (PDF-format)