Appendix 



V. IMMUNOFLUORESCENCE BUFFERS AND MOUNTING FLUIDS 



Some immunofluorescence techniques utilize pliosphate buffers and others call for carbon- 

 ate buffers. Both systems are recommended in this manual and presented below. 



A. Phosphate Reagents 



1 . Phosphate Buffered Saline (PBS), pH 7.6, 0.01 M: 



a. Concentrated Stock Solution (pH will not be 7.6); 



Naj HPO4 (anhydrous; reagent grade) 12.36 gm 



NaH, PO4 -H, O (reagent grade) 1 .80 gm 



NaCl (reagent grade) 85.00 gm 



Distilled water to make final volume 1 liter 



Dibasic salt dissolves much more readily in water at 37°C 

 than at :5°C. 



b. Working Solution (pH 7.6; 0.01 M buffer; 0.85% NaCl): 



Concentrated stock solution 100 ml 



Distilled water to make final volume 1 liter 



:. Phosphate Buffered Saline (PBS), pH 7.2: 



0.15 M KH, PO4 24 ml 



0.15 MNa2HP04 76 ml 



0.85% NaCl 100 ml 



3. Buffered Glycerol Mounting Fluid, approx. pH 9.0: 



Glycerol, reagent grade 90 ml 



0.2 M Na. HPO4 10 ml 



B. Carbonate Reagents 



1 . Carbonate-Bicarbonate Buffer, pH 9.0, 0.5 M: 



a. 0.5MNa2CO3 5.3 g/ 100 ml of distilled water 



b. 0.5 M NaHCOj 4.2 g/100 ml of distilled water 



c. Mix 4.4 ml of solution (a) with the 100 ml of solution (b). Theoretically, a pH 

 of 9.0 should result from this mixture; however, as much as 17.0 ml of solution 

 (a) may have to be added to the 100 ml of solution (b). 



2. Glycerol Mounting Fluid: 



a. 0.5 M Carbonate-bicarbonate buffer, pH 9.0 1 part 



b. Neutral glycerol, reagent grade 9 parts 



c. Combine and mix by stirring (do not shake). 



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