Antifade

Mounting mount sections in aqueous medium or balsam for brightfield microscopy or in antifade medium for fluorescence microscopy (see Sect. 3.2.2). Notes. A11 incubations are at room temperature unless otherwise noted. Nuclear dyes (DAPI, Hoechst 33342 and Propidium Iodide) supplied as lyophilized solids are usually reconstituted in methanol. The stock solutions (5 mg/ml) are stable for many years when stored frozen at < 20°C and protected from light. Before use, the stock solution is further diluted in PBS to the final concentration of 5 pg/ml. 

Mounting mount sections in antifade medium for fluorescence microscopy (see Sect. 3.2.2). 

Mounting mount sections in antifade medium for fluorescence microscopy. 

Antifade agents (free-radical scavengers) in aqueous mounting media prevent photobleaching of fluorophore labels under exposure to excitation light. 

G. Bock, M. Hilchenbach, K. Schaenstein, and G. Wick, Photometric analysis of antifading reagents for immunofluorescence with laser and conventional illumination sources,... 

Florijn RJ, Slats J, Tanke HJ, Raap AK. Analysis of antifading reagents for fluorescence microscopy. Cytometry 1996 19(2) 177-182. 

Add an aqueous mounting medium or an antifade solution, mount a coverslip and analyze using fluorescent microscopy with a fluorescein filter. 

Glycerol-gel mounting medium containing an antifading agent (Vectashield Vector). 

Use the relevant visualization steps described in Subheading 3.3. for IGSS, IPO, or lAP, in sequential reactions. Sections stained for immunofluorescence should be mounted with a glycerol-gel solution containing an antifading agent such as Vectashield (Vector). 

Antiavidin antibody solution (Vector Labs) 5 pg/mL in PBS with 1% Tween-20. Propidium iodide (Sigma, St. Louis, MO) counter stain 1 pg/mL in PBS. Antifade mounting medium 100 mg p-phenylenediamine dihydrochloride in 10 mL PBS adjust to pH 9 (see Note 3). 

Stain slides with Hoechst 33258 for 10 min, and mount in antifade solution. 

The sections are mounted in a suitable antifade medium such as VectaShield (Vector Laboratories, Burlingame, CA). 

ProLong Gold antifade reagent with 4 ,6-diamidino-2-phenylindole DAPI (Invitrogen, cat P36931)... 

After the final wash, the coverslips are drained of excess liquid and are mounted by inverting onto 5 pil Mowiol mountant (Cal-biochem) containing p-phenylenediamine (1 mg/ml) as an antifading agent. 

ANTIFADE + PI +DAPI Add to antifade (Vector Labs, Burlingame, CA) solution, 0.3 pg/mL Propidium iodide (PI) 0.1 pg/mL 4, 6-diamidine-2 -phenylindole dihydrochloride (DAPI). 

Dehydrate and mount slides in Antifade with PI and DAPI (see Note 8). 

Prepare antibleach medium with fluorescent counterstain 90% glycerol and 2.5% l,4-diazabicyclo[2.2.2]oclane (DABCO antifade. Sigma) in IX PBS to which either 1 mg/ml 4,6-diamidino-2-phenylimdole dihydrochloride (DAPI, Sigma) or 5 mg/ml propidium iodide is added. 

WO 99/14245 03/1999 Leupin et al.l Procter Gamble Laundry detergent compositions containing cellulosic-based polymers to improve fabric appearance and integrity Laundering of fabric/textiles with the additive leads to overall improvements in fabric appearance, pill/fuzz reduction, antifading properties, improved abrasion resistance, and enhanced softening... 

Antifade mounting medium such as Fluoromount G (Electron Microscopy Sciences, Hatfield, PA). 

Mount approximately 30 pL of the cell suspension on an adhesive coated coverslip (see Note 4) and mount on slide with antifade mounting medium. Examine with a fluorescent microscope equipped with the appropriate filters (see Notes 6-9). 

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