Table I.

Double‐Staining Protocols with PAG

Four‐step staining

(two primary antibodies, both able to bind PAG directlya)

First primary antibody/PAG‐1 ‐GA‐b Second primary antibody/PAG‐2

Five‐step staining

(one primary antibody requiring a bridging antibody, the second, from a different species able to bind PAG directly)

First primary antibody/Bridging antibody/PAG‐1 ‐GA‐ Second primary antibody/PAG‐2

OR First primary antibody/PAG‐1 ‐GA‐ Second primary antibody/Bridging antibody/PAG‐2

Six‐step staining

(two primary antibodies from different species, both requiring a bridging antibody)

First primary antibody/Bridging antibody/PAG‐1 ‐GA‐ Second primary antibody/Bridging antibody/PAG‐2

(two mouse primary antibodies, the first requiring a rabbit anti‐mouse IgG‐bridging antibody)

First primary antibody/Bridging antibody/PAG‐1 ‐GA‐ Isotype‐matched blocking antibodyc ‐GA‐ Second primary antibody/PAG‐2

^aStrong protein A‐binding antibodies include (among others) rabbit antibodies, human IgG1 and 2 (not IgG3) and some mouse IgG 2a, 2b. Antibodies from rat, goat, or sheep do not generally bind to protein A.

^bGA denotes the fixation to inactivate protein A‐binding sites. This consists of 5–10 min in 1–2% glutaraldehyde, followed by washing in PBS, quenching in 50‐mM glycine buffer, as well as a preincubation in BSA‐containing blocking buffer.

^cSame isotype as the first primary antibody.