Immunohistology of mouse spleens

doi:10.1038/nprot.2007.40

Method Article

Immunohistology of mouse spleens

https://doi.org/10.1038/nprot.2007.40

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Genetics

Biological techniques

Immunohistology

mouse

spleen

This protocol was used in our Nature Immunology paper.

OCT compound embedding medium (Sakura, Finetek, Torrance, CA)

Liquid nitrogen

Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA)

Coverslips (Fisher Scientific, Pittsburgh, PA)

Acetone (histological grade, Sigma)

PBS + 5% fetal calf serum

Anti-CD3-phycoerythrin (Pharmingen)

Anti-B220-FITC (Pharmingen)

Anti-peanut-agglutinin-biotin (US biological)

Streptavidin-cyanin 5.5 (Pharmingen)

PBS + 0.05% Tween 20 (Sigma)

Gelmount (Biomeda, Foster City, CA)

Harvest of mouse spleens and preparation of splenic sections

1 Harvest mouse spleens, embed them in OCT compound embedding medium and freeze in liquid nitrogen.

2 Cut 7 µm sections and mount them on Superfrost Plus slides.

3 Incubate slides in humidified chambers overnight at 4 ˚C and dry them, then fix them in acetone at 4 ˚C for 10 min and block them with PBS + 5% fetal calf serum at 25 ˚C for 1 h.

Antibody staining and microscopy

4 Stain blocked sections overnight at 4 ˚C with a combination of anti-CD3-phycoerythrin, anti-B220-FITC, and peanut-agglutinin-biotin, all diluted in blocking buffer (all the antibodies need to be titrated for optimal results; dilution 1:200 – 1:1000) (1).

5 Wash the slides 3x with PBS + 0.05% Tween 20. Stain sections with streptavidin-cyanin 5.5 (dilution 1:100) at 25 ˚C for 1 h.

6 Wash the slides 3x with PBS + 0.05% Tween 20, dry them and mount them with Gelmount.

7 Capture images at x10 magnification with a Zeiss Axioplan microscope.

2 d

Anzelon, A. N., Wu, H. & Rickert, R. C. Pten inactivation alters peripheral B lymphocyte fate and reconstitutes CD19 function. Nat Immunol 4, 287-294 (2003).
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Engel, P. et al. Abnormal B lymphocyte development, activation, and differentiation in mice that lack or overexpress the CD19 signal transduction molecule. Immunity 3, 39-50 (1995).
Gallagher, E. D., Xu, S., Moomaw, C., Slaughter, C. A. & Cobb, M. H. Binding of JNK/SAPK to MEKK1 is regulated by phosphorylation. J Biol Chem 277, 45785-45792 (2002).
Gallagher, E. D., Gutowski, S., Sternweis, P. C. & Cobb, M. H. RhoA binds to the amino terminus of MEKK1 and regulates its kinase activity. J Biol Chem 279, 1872-1877 (2004).
Rush, J. S., Hasbold, J. & Hodgkin, P. D. Cross-linking surface Ig delays CD40 ligand- and IL-4-induced B cell Ig class switching and reveals evidence for independent regulation of B cell proliferation and differentiation. J Immunol 168, 2676-2682 (2002).

Supported by the National Institutes of Health (ES04151 and ES06376 to M.K.), the American Cancer Society (M.K.) and the Leukemia and Lymphoma Society of America (T.E.; SCOR grant 7332-06).

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Immunohistology of mouse spleens

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