66 X.-Q. Zhang et al. purified from porcine liver cytosol and cloned from a human kidney genomic DNA library. It was named phosphohistidine phosphatase (PHPT1), since no phos- phoprotein substrates were described at the time (7). This enzyme was independently identified by Klumpp et al. (8) in rabbit liver. The latter group named the enzyme protein histidine phosphatase. The full physiological function of PHPT1 remains to be elucidated, although it has been suggested that the enzyme is involved in the regulation of signalling via G proteins (9) and regulation of ATP-citrate lyase (10). Recently Srivastava et al. (11) described that K channel KCa3.1 is dephosphorylated by PHPT1, thereby affecting CD4 T cell function that could have an impact on autoimmunity. PHPT1 has been shown to be rather conserved between species. To increase the knowledge of the role of PHPT1, we have studied, in the present work, the expression of the enzyme on protein level by an immunohistochemical approach. An earlier investigation of a Northern blot of human tissue using a PHPT1 cDNA as probe (7) showed the expression of a 0.6-kb mRNA mainly in the heart and skeletal muscle, whereas the expression in liver was lower. Multiple organs from mouse were in- cluded in the present study. Since PHPT1 is homologous to a sex-regulated protein in Drosophi- lae, also testis was included. Organs of mouse embryo from different stages of development were investigated as well. The PHPT1-specific antibodies employed in this study were in parallel used in the Swedish human proteome resource project (HPR project, http://www.proteinatlas.org/in- dex.php). These latter data are also discussed in this paper. Materials and methods Tissue samples and histological pretreatment Tissue samples were obtained from adult mice (4 weeks to 8 months old) and E14.5 mouse embryos. The specimens were collected from B6CBAF1 and C57BL/6J mice from Charles River Laboratories (Uppsala, Sweden). All animal experiments had been approved by the Uppsala animal ethics com- mittee. Organs were fixed in 4% paraformaldehyde in 100 mM sodium phosphate/100 mM NaCl, pH 7.5 (PBS). The samples were then embedded in paraffin and sectioned to 6 mm thickness and stored at 48C. The slides were heated for 30 min at 608C the same day the immunohistochemistry was per- formed. Prior to the immunohistochemical treat- ment the sections were deparaffinized in xylene for 2 5 min and rehydrated in decreasing concentra- tions of ethanol: 100%, 95%, 70%, for 2 2 min in each concentration, after which the sections were washed twice in PBS for 5 min. Antibodies The primary antibody was raised by Innovagen in rabbit against whole recombinant human PHPT1 that was prepared as described by Ek et al. (7). The rabbit immune serum was affinity-purified against a PHPT1-specific peptide (101 115) bound to CNBr- Sepharose (Innovagen). Peptide 101-115 was cho- sen since it is essentially identical in several species including mice and humans. Biotinylated goat anti- rabbit immunoglobulin G (IgG), purchased from DAKO, was used as secondary antibody. Immunohistochemistry The slices were proteinase K (Roche)-treated (10 mg/ mL in 50 mM Tris/5 mM EDTA, pH 7.5) in order to minimize obstructions that could prevent the bind- ing of the antibody. The tissues were subjected to this treatment for 20 min at room temperature. The proteinase was removed by immersion of the sec- tions in PBS for 2 5 min. To reduce background staining, additional pre- treatment was conducted by immersing the slides in 3% (v/v) H2O2 in PBS for 5 min at room tempera- ture followed by PBS for 2 2 min. Thereafter the sections were blocked in a mixture of 3% (w/v) bovine serum albumin (BSA) and 5% (v/v) goat serum in PBS for 30 min at room temperature. The blocking solution was removed by PBS for 2 5 min. The primary, affinity-purified antibody, with an original concentration of 0.36 mg/L, and diluted in PBS 1/1000 or 1/100, was added, and the slides were incubated at 48C for 16 h. In absorption control experiments the 1:1000 diluted primary antibody was mixed with 10 mM peptide 101-115 and 3% BSA just before incubation. Preimmune serum was diluted 1/1000 in PBS, when used. The slides were rapidly warmed at room tem- perature. After this, the primary antibody was removed with PBS containing 0.1% (v/v) Tween 20 for 5 3 min (shaking). Secondary antibody, diluted 1/1000 in PBS with 3% BSA, was added, and the slides were incubated for 2 h at room temperature. The secondary antibody was then removed with PBS in 0.1% Tween 20 for 5 3 min (shaking). The slides were incubated at room temperature for 45 min with avidin-biotin-horseradish perox- idase-complex, diluted 1/1000 in PBS. After wash- ing with PBS/0.1% Tween 20 for 5 3 min, the