A practical guide to in situ and ex situ characterisation of arabinogalactan proteins (AGPs) in fruits

Table 4 Reagents and equipment necessary for immunocytochemical techniques

Immunofluorescence labelling	Immunogold labelling	SDS-PAGE Western blotting	ELISA
Reagents
PBS	PBS	PBS	PBS
BSA	BSA	BSA	BSA
Paraformaldehyde	Uranyl acetate	12.5% Resolving gel*	NaOH
Glutaraldehyde	Reynolds reagent	Stacking gel*	PNPP
NaOH	Paraformaldehyde	10% APS
Ethanol	Glutaraldehyde	Sample buffer*
Resin	NaOH	Running buffer*
	Ethanol	Transfer buffer*
	Resin	TBST*
		AP buffer*
		BCiP*
		NBT*
		DMF
Antibodies
Primary antibody diluted in BSA (1:50)	Primary antibody diluted in BSA (1:10)	Primary antibody diluted in PBS (1:500)	Primary antibody diluted in PBS (1:20)
Secondary antibody conjugated with a fluorochrome diluted in BSA (1:200)	Secondary antibody conjugated with gold nanoparticles diluted in BSA (1:50)	Secondary antibody with AP diluted in PBS (1:1000)	Secondary antibody with AP diluted in PBS (1:500)
Equipment
Vacuum pump	Vacuum pump	Electrophoresis apparatus	Microplate washer
Gelatine capsules	Gelatine capsules	Wet transfer module	Microplate shaker
Ultramicrotome	Ultramicrotome	Laboratory shaker	Microplate reader
Glass knife	Diamond knife	Nitrocellulose membrane
Poly-L-lysine coated glass slides	Nickel grids with formvar film	Scanning machine
Wet chamber	Petri dishes
Hydrophobic pen	TEM
CLSM

^*Resolving gel—12.5% (33.28 mL of acrylamide, 25.12 mL of distilled water, 20 mL of 1.5 M Tris pH 8.8, 0.8 mL of 10% SDS)
^*Stacking gel (5.44 mL of acrylamide, 21.76 mL of distilled water, 4 mL of 1 M Tris pH 6.8, 0.32 mL of 10% SDS, bromophenol blue)
^*Sample buffer (100 µL of Tris pH 6.8, 480 µL of 10% SDS, 480 µL of 0.5 M DTT, 240 µL of 100% glycerol)
^*Running buffer (add 14.4 g of glycerine, 3 g of Tris Base, and 1 g of SDS to 1 L of distilled water)
^*Transfer buffer (add 72.06 g of glycine, 15.14 g of Tris Base, and 20 mL of 10% SDS to 500 mL of distilled water)
^*TBST (add 2.423 g of Tris Base, 8 g of NaCl, and 1 mL of Tween 20 to 1 L of distilled water and establish pH 7.6)
^*AP buffer (add 12.11 g of Tris Base, 5.84 g of NaCl, and 1.01 g of MgCl₂ to 1 L of distilled water and establish pH 9.5)
^*BCiP (add 4 mg of BCiP to 1 mL of distilled water)
^*NBT (add 9 mg of NBT to 0.3 mL of distilled water and 0.7 mL DMF)

ISSN: 1746-4811