Staining buffer

Dilute the resultant primary antibody Fab fragment complexes in staining buffer containing 10 20% normal serum from the same species as the primary antibody to give the concentration of about 1 10 pg/ml of the primary antibody, and incubate for 15 30 min at room temperature to block unbound labeled monovalent Fab fragments. ... 

Further dilute (if required) the resultant primary antibody Fab fragment complexes to optimal working concentration (usually about 1 5 pg/ml) in staining buffer containing 10% normal serum and then apply to the sample for 30 60 min at room temperature and proceed further with your standard immunostaining protocol. 

Incubate specific mouse monoclonal primary antibody with FITC -conjugated mouse IgG specific Fab fragments at a ratio of 1 2 (weight for weight, based on concentration data supplied by manufacturers) in a small volume (e.g., in 10 pi or more, typically 1 pg of primary antibody in 10 pi) of staining buffer in a microcentrifuge tube for 20 30 min at room temperature. 

Dilute the resultant complexes in staining buffer containing 10% normal mouse serum to give the working concentration of 5 pg/ml of primary mouse IgG, and incubate for 10 20 min at room temperature to block unbound Fab fragment paratopes. 

NTB/BCIP 0.33 mg/mL nitroblue tettazolium salt and 0.16 mg/mL 5-bromo-4-chloro-3-indolylphosphate in stain buffer. 

Add four drops of a freshly prepared solution of NTB/BCIP in stain buffer, and incubate at 37°C. Examine for development of blue stain after 90 min. If color is detected, stop the reaction by rinsing slides in T-E, pH 7.4. If blue color is not detected, resume incubation at 37°C, and reexamine every 10 min. 

AP staining buffer 0. lMNaCl, 0.lMTris, pH 9.5, 50 mMMgCl2. Dissolve Tns and NaCl in distilled water (4/5 final volume), adjust pH to 9.5 with HC1. Make up a small stock solution of 4M MgCl2 in distilled water (e.g., 50 mL), and add the appropriate amount to the staining buffer (12.5 mL/L). Make up to the final volume with distilled water and store at room temperature. Stable for 6 mo. 

Vortex mix the NBT and BCIP stock solutions, and add 100 pL of each to 50 mL AP staining buffer When pipeting BCIP, it is useful to snip off the end of the pipet tip to widen its bore. 

Rinse the blot briefly in staining buffer (15s) before submerging it in the staining solution. Cover the container in aluminum foil to block out any light. AP reactions can develop quite slowly, and it may take an hour or more before an acceptable... 

Labeling mixture For each reaction, make up 250 pL staining buffer, 0 1 pg antidigitoxin-FITC conjugated antibody (Boehnnger Mannheim). Use immediately. 

Resuspend the cells in 500 pL staining buffer + 5% (w/v) Marvel. Centrifuge at 170g for 5 min. Discard the supernatant. 

Antibody staining buffer PBS containing 0 5% (v/v) Tween-20 and either 1% (w/v) BSA (for direct antibody conjugates), or 0 5% (v/v) normal goat serum when a second antibody is used. 

Resuspend pellet in 90 pL of antibody staining buffer containing BSA (Section 2), and add 10 pL (see Note 7) of MAb The recommended antibodies are PC 10-FITC (DAKO F863) and Ki-67-FITC (DAKO F788). The appropriate directly conjugated isotypic control antisera are also available from DAKO The suspensions are incubated for 1—2 h at room temperature and protected from light... 

Appendix Stain Buffer Formulations and Enzyme Activity Stain Protocols... 

Gently rock the staining trays on occasion to ensure that the stain buffer is evenly distributed over the gel. Gels should be incubated in the dark at 37° unless otherwise noted. Monitor the gels closely and stop the reaction (by pouring off the stain solution and adding water or fixative) when the resolution of the bands is optimal. 

Sample staining buffer (0.1% (w/v) NaNj and 1.0% (w/v) bovine serum albumin in Hanks balanced salt solution)... 

Resuspend the formulation-stimulated dendritic cells with staining buffer and aseptically prepare a single-cell suspension. 

Wash the cells two to three times with sample staining buffer. 

Staining the Actin Cytoskeleton 1. Intracellular buffer (2x). 280 mM KCl, 2 mM MgCl, 4 mM EGTA, 40 mM Hepes of pH 7.5, 0.4% low endotoxin albumin from human serum (Sigma) see Note 5). 2. Fixation buffer (2x). 640 mM sucrose, 7.4% formaldehyde (Sigma) in 2x intracellular buffer store at 4°C (tee Note 6). 3. Stain buffer. 0.2% triton, 2 pl/ml rhodamine phalloidin (Invitrogen) in lx intracellular buffer (rccNote 7). 

Fig. 6. Staining the actin cytoskeleton. Add 2x fixation buffer to plated cells and tlx for 20 min at 4°C. Remove fixation buffer and replace with stain buffer for 20 min protect from light. Shown Is an example of an FIL-60 cell stained with rhodamlne phalloldin visualized with structured Illumination microscopy.

Permeabilize cells with 125 pi stain buffer and protect from light for 20 min (Fig. 6). 

Incubate the sections overnight at room temperature in staining buffer containing 1 mg/mL X-gal, 0.02% sodium deoxycholate and 0.01% Nonidet P-40 (NP-40)... 

The diluted X-gal in staining buffer can be reused several times. 

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