114 Morioka et al. muscle conventional myosins, which have been suggested to be active in cytokinesis [8, 14], cell movement [1, 22], chemotaxis [2, 6], the postmitotic spreading of cells [4], cell shape [6, 15, 24], stabilization of the actin cytoskeleton [29], incorporation of sperm [23] and enucleation of erythroid cells [26]. Jazwinska et al. studied the distribution of skin and follicular antigens as they cross-reacted with antibodies raised against skeletal muscle myosin heavy chains [10]. In our investigation, we used antibodies against muscle and nonmuscle myosin heavy chains to study the localization of muscle and nonmuscle myosins in the skin and hair follicles based on the authentic antigen-antibody reactions. II. Materials and Methods Preparation of samples Sprague-Dawley rats were obtained from Nippon SLC Inc. (Hamamatsu, Japan). Animals 5 to 7 days old were used for all experiments. The skin tissue used for immunoblotting was cut into small fragments in the presence of 100-fold diluted protease inhibitors (the undiluted mixture is a product of Wako Pure Chemical Industries Ltd., Osaka, Japan; code 160-19501) dissolved in phosphate-buffered saline, frozen quickly in liquid nitrogen, and stored at 80C until use. The tissues for immunohistochemical staining were fixed in 10% formalin neutral buffer solution (Wako) for 24 hr at 4C, dehydrated, and embedded in Shandon Histoplast paraffin (Thermo Electron Corp., Pittsburgh, PA). The blocks were sectioned at 4 µm thickness with a Leica RM2135 microtome (Leica Microsystems AG, Wetzler, Germany). Primary antibodies Mouse anti-chicken gizzard actin monoclonal antibody (clone C4) was purchased from MP Biochemicals Inc., Aurora, OH. Mouse anti-human skeletal muscle myosin heavy chain monoclonal antibody (clone A4) was purchased from Upstate Corp., Charlottesville, VA. Rabbit anti-human platelet myosin polyclonal antibody (BT-561) was purchase from Biomedical Technologies Inc., Stoughton, MA. C4 and A4, both consist of mouse ascites and additives, were diluted to 1:100 or 1:200 by 0.1%BSA in PBS, while BT-561 (a package of purified IgG solution) was diluted to 1:20 by the same buffer for histochemical study. For immunoblotting, C4, A4, and BT561 were diluted to 1:500, 1:500 and 1:100, respectively. Immunoblotting Stored frozen samples were struck with a stainless steel rod (SK200, Tokken Inc., Chiba, Japan) and crushed into powdery pieces. The temperature was kept below freezing during this operation. The powdery pieces were then heated at 95C for 2 min in 125 mM Tris-HCl (pH 6.8), 4.3% sodi- um dodecyl sulfate, 10% 2-mercaptoethanol, 30% glycerol, and 0.01% bromophenol blue solution. Polyacrylamide gra- dient gels (4 to 20%) for electrophoresis were purchased from Daiichi Pure Chemicals Co., Ltd. (Tokyo, Japan). Immunoblotting ABC-POD kits for mice and rabbits were purchase from Wako; the experiments were performed according to the manufacturer’s manual with Wako supplies except for the blotting buffer (EzBlot), which was obtained from Atto Corp. (Tokyo, Japan). EzBlot is composed of three different buffers for anode, membrane gel, and cath- ode, respectively, to facilitate the transfer. We transferred the proteins to polyvinylidenefluoride (PVDF) membrane at 2 mA/cm2 for 60 min. Marker proteins were obtained from Daiichi Pure Chemicals. Immunohistochemistry For immunohistochemical experiments, we used Histofine Simple-Stain Rat MAX-PO (MULTI) as the sec- ondary antibody, a product of Nichirei Co., Tokyo, Japan. The experiment was carried out according to the procedure described by the manufacturer. MAX-PO consists of a poly- mer conjugated with Fab secondary antibody and peroxi- dase. Localizations of the complex were visualized by 3-3'- diaminobenzidine (DAB). Incubation of sections with pri- mary antibodies prepared as described before was carried out for 24 hr at 4C and then with MAX-PO for 30 min at room temperature. Sections colored by DAB were post- stained briefly with methylgreen-pyronine [17]. In addition to the immunoperoxidase staining, some sections were stained with hematoxylin and eosin for histological exami- nations. An Olympus BX-51 microscope equipped with a Camedia C-3040 digital camera system was used to take photographs. Immunofluorescent staining was carried out using fluorescein-4-isothiocyanate (FITC)-conjugated donkey anti-mouse IgG antibody and Rhodamine Red-X-conjugated donkey anti-rabbit IgG antibody, which were obtained from Jackson Immunoresearch Laboratories, Inc. West Grove, PA. The former was subjected to 200- and the latter to 500- fold dilution. For nuclear counterstaining, they were then mixed with 2 g/ml of 4',6-diamidino-2-phenylindole dihy- drochloride (DAPI). The incubation with the secondary anti- bodies was performed for 30 min at room temperature. III. Results Immunoblotting Both the anti-skeletal muscle myosin II heavy chain antibody (Fig. 1A: lane 1, 2) and anti-nonmuscle myosin II heavy chain antibody (Fig. 1A: lane 3, 4) were shown to react with proteins corresponding to the molecular weight of myosin heavy chain. Anti-actin antibody reacted with the protein corresponding to actin (Fig. 1B). Samples in Figure 1A1, A3, and B1 were obtained from the dorsal skin and those in Figure 1A2, A4, and B2 were from the mystacial pad. The reactions were shown to be largely specific, though minor bands around 70 kDa were detected in the reactions of the antibody against anti-skeletal muscle myosin heavy chain (Fig. 1A1 and A2). Significant differences in the pat- terns in dorsal skin versus mystacial pad samples were not observed.