Seven Hills Bioreagents

Specimen preparation:

Dewax sections through 3 to 4 changes xylene and rehydrate through descending alcohol to water. Soak in PBS for 5 min. Quench endogenous peroxidase for 15 min at room temperature with 1.5% hydrogen peroxide in methanol.


  • Antigen recovery/retrieval, (required for any nuclear antigen): Immerse sections in 10 mM sodium citrate, pH 6.0, and heat in a microwave for 15 min at 90oC.
  • Blocking: Block sections in 2-4% (unless otherwise indicated) normal serum* in PBS + 0.2% Triton X-100 (normal serum block) for 2 hr at room temperature.

    *NOTE: serum should be from the same source that the secondary antibody is derived from.

Staining procedure:

Incubate samples overnight at 4oC with the primary antibody diluted in normal serum block (2-4% normal goat serum in PBS/0.2% Triton X-100) at the dilution indicated. Wash sections 5 times for 5 min each in PBS containing 0.2% Triton X-100. Incubate 30 min at room temperature with biotinylated anti-rabbit secondary antibody diluted 1:200 in blocking solution.

To detect antigen:antibody complexes use a Vectastain ABC Peroxidase Elite Rabbit IgG kit as follows: Incubate sections for 30 min at room temperature with avidin/biotin/peroxidase complex (ABC kit from Vectastain) diluted in blocking solution, then NiDAB in 0.1 M acetate buffer for 4 min at RT, then Tris cobalt for 4 min at RT. Counterstain with nuclear fast red for 2 min at RT.


Controls include 1) preimmune and/or nonimmune rabbit sera or rabbit IgG 2) omission of the primary antibody to check for endogenous biotin and peroxidase activity, as well as nonspecific binding of the secondary antibody.


Sign up to receive our newsletter, updates and our latest product releases.

We will not share, rent, or give your name or email address to anyone else. Please see our privacy policy if you have any questions.

Recently Published Articles

Chen G, Korfhagen TR, Karp CL, Impey S, Xu Y, Randell SH, Kitzmiller J, Maeda Y, Haitchi HM, Sridharan A, Senft AP, Whitsett JA.
Foxa3 induces goblet cell metaplasia and inhibits innate antiviral immunity. Am J Respir Crit Care Med. 2014 Feb 1;189(3):301-13.

Balli D, Ustiyan V, Zhang Y, Wang IC, Masino AJ, Ren X, Whitsett JA, Kalinichenko VV, Kalin TV.
Foxm1 transcription factor is required for lung fibrosis and epithelial-to-mesenchymal transition. EMBO J. 2013 Jan 23;32(2):231-44.

Dong M et al.
ERdj4 and ERdj5 are required for endoplasmic reticulum-associated protein degradation of misfolded surfactant protein C. Mol. Biol. Cell 2008;19:2620-2630.

Stahlman MT et al.
Expression of ABCA3 in developing lung and other tissues. J Histochem Cytochem 2007;55:71-83.