Skip to main content
. 2007 Jul 4;27(27):7344–7360. doi: 10.1523/JNEUROSCI.0873-07.2007

Table 1.

Reagents used for PVH immunocytochemistry

Reagent Antigen/conjugate Host Type Source Catalog no. Lot no. Incubation Dilution
Immunoperoxidase reagents
    Primary p44/42 MAP kinases a Rb Polyclonal IgG CST 9101 12, 13 b 72 h, 4°C 1:1000
    Secondary Anti-rabbit IgG Gt Biotinylated KPL 71-00-30 Varies 2 h, RT 1 part in 3
    Conjugate Streptavidin HRP conjugated KPL 71-00-38 Varies 1–1.5 h, RT 1 part in 2
    Chromagen DAB HistoMark Enhanced Black kit KPL 54-75-00 Varies 4 min, RT 12% (v/v) c
Immunofluorescence reagents
    Primary p44/42 MAP kinases a Ms Monoclonal IgG CST 9106 8 d 72 h, 4°C 1:1000
    Secondary Anti-mouse IgG e Dk Cy3 conjugated Jackson 715-165-150 65442 8–12 h, 4°C 1:500
    Primary CRH Rb Polyclonal IgG W. Vale C-70 72 h, 4°C 1:4000
    Secondary Anti-rabbit IgG f Dk Biotinylated Jackson 711-065-152 Varies 6–8 h, RT 1:500
    Conjugate Streptavidin Alexa 488 conjugated Invitrogen S-11223 37350A 8–12 h, 4°C 1:500

Rb, Rabbit; Gt, goat; Ms, mouse; Dk, donkey; CST, Cell Signaling Technology (Danvers, MA); KPL, Kirkegaard & Perry Labs (Gaithersburg, MD); Jackson, Jackson ImmunoResearch (West Grove, PA). W. Vale is at The Salk Institute (La Jolla, CA). Invitrogen is located in Carlsbad, CA.

aThe specific epitope recognized is for phosphoryl modifications of human p44/42 MAP kinases at Thr202 and Tyr404 (Thr183 and Tyr185 in rat ERK2). These antibodies recognize single bands on immunoblots, corresponding to ERK1 and ERK2, respectively (according to supplier's data).

bThese lots were assayed by the supplier in February 2002 and July 2002, respectively.

cFor 50 ml of solution, 5 ml of the kit's Enhanced Buffer and 1 ml of its DAB solution were used (thus, 6 ml for every 50 ml, or 12% final concentration). The remaining reagents were as follows: 1 ml of H2O2 and 43 ml of distilled H2O.

dThis lot was assayed by the supplier in August 2004.

eThis secondary was rabbit serum adsorbed.

fThis secondary was mouse serum adsorbed.