Subcellular distribution of the 1,4-dihydropyridine receptor in rabbit skeletal muscle in situ: an immunofluorescence and immunocolloidal gold-labeling study.

Monocional Antibodies to the 1,4-Ih'hydropyridine Receptor The mAbs to the 170,000-D (t~l) and the 52,000-D (~) subunits of the 1,4-dihydropyridinereceptor from rabbit skeletal muscle were prepared and characterized as previously described (24, 25). Immunoblotting was per- formed according to the methods of Towbin (42) using 5% non-fatdry milk in PBS (BLOTTO) for blocking and washing of the immunoblots as de- scribed (15). Determination of the immunoglobulinclass of the rnAbs was carded out by immunoditfusionaccording to the procedure of Ouchterlony (29). Each of the mAbs was immunoditfusedagainst monospecific antibod- ies to mouse IgGi, IgG2a,IgG2b,IgG3, and IgM. mAb IIID5 to the t~t-sub- unit of the 1,4-dihydropyridine receptor used for immunoelectron micros- copy was purified from the mouse ascites fluid by DEAE-Affi Gel Blue chromatography (1). Monoclonal Antibodies to the CaZ/-ATPaseof the Sarcoplasmic Reticulum mAb IID8 to the Ca2+-ATPase from canine cardiac sarcoplasmic reticulum and mAb IIHll to the Ca2+-ATPaseof rabbit skeletal sareoplasmic reticu- lure used in this study were prepared, purified, and characterized as previ- ously described (18). Preparation of Skeletal Muscle Extract, Triads, and Transverse Tubule Membranes The extract represents the supornatant obtained from tissue homogenates during the preparation of triads. Triads were prepared as described by Mitchell et al. (28) with slight modification (39). Transverse tubular mem- branes were prepared by the method of Rosemblatt et al. (34). Protein con- centrations were determined by the method of Lowry (26) as modified by Peterson (31)using BSA as a standard. SDS-PAGE on 3-12% gradiem gels was performed by the method of Laemmli (23). Dissection, Fixation, and Sectioning Fixed and unfixed bundles of skeletal muscle fibers from rabbit gracilis and psoas muscle were prepared as previously described (20). Briedfly, bundles of myofibers from rabbitgracilis or psoas muscle were dissected and quickly frozen in liquid nitrogen-cooled isopentane. Bundles of myofibers to be fixed were dissected from rabbit gracilis or psoas muscle and immediately tied to applicator sticks (for chemical fixation) or stainless steel loops (for cyrofixation) at 100-120% of their rest length and allowed to recover for 30 rain in a modified Krebs-Henseleitbuffer (145 mM NaCI, 2.6 mM KCI, 5.9 mM CaCI2, 1.2 mM MgSO4, 25 mM NaI-ICO3, and 10 mM glucose, satu- rated with a mixture of 95% 02 and 5% C02). The bundles of myofibers to be used for immunofiuorescence studies were fixed for 3 h in ice-cold 2% paraformaldehyde in 0.1 M sodium cacodylate (pH 7.4). Sucrose infu- sion, storage, and cryosectioning (6-8 ttm) were carried out as previously described. The bundles of myofibers to be used for immunocolloidal gold labeling were cryofixed, freeze-dried, and low temperature embedded in Lowicryl K4M as previously described (Procedure II [17]) except that 10 rainbefore freezing, the Krebs-Henseleitbuffer was changed to also include 4% polyvinyl pyrrolidone (Sigma Chemical Co., St. Louis, MO) as a cryoprotectant (19). Briefly the bundles of myofibers were cryofixed using the "Gentleman Jim" freezing device (Ted Pella, Tustin, CA) as described by Philips and Boyne (32). The cryofixed tissue was then quickly transferred to liquid nitrogenand storedovernight. Freeze-dryingwas carried out at low temperature in a glass cryosorption pump (5, 27). Infiltration and embed- ding of the cryofixed, freeze-dried tissue in ~icryl K4M was performed according to the procedure of Chiovetti et al. (5, 6) as modified by Jorgensen and McGuffee (17). Thin sections (60-80 nm) were collectedon nickelgrids coated with formvar.

Immunofluorescence Labeling

Immunofluorescence staining of cryosections from unfixed rabbit gracilis was carded out as previously described (20). The sectionswere first labeled with one of the following rnAbs: HD8 to Ca2+-ATPase of canine cardiac sarcoplasmic reticulum; HHll to Ca2+-ATPaseof rabbit skeletal sarcoplas- mic reticulum; HID5 and IICI2, to the ~l-subunit of the 1,4-dihydrol~ri- dine receptor; and VD21 to the/3-subunitof the 1,4-dihydropyridine recep- tor. The secondary antibody was F(ab')2 fragments of affinity-purified goat anti-mouse IgG conjugated to ~ (CooperBiomedical Inc., Malvern, PA). It was used at a dilution of 1:20. The fluorescence photographs were taken with a Zeiss photomicroscope provided with an Epi-fluorescence at- tachment and a phase-contrast condenser using Kodak Tri-X pan film. Immunotluorescence staining of cryosections from fixed rabbit gracilis muscle were carded out as described for cryosections from unfixed rabbit gracilis muscle, except that the secondary antibody used above was sub- stituted with a two-step incubation described below. After incubation with the primary antibodies, cryosections were washed in PBS (pH 7.4) and then incubated for 30 rain with a 1:25 dilution of biotin conjugated to affinity- purified F(ab')2 fragments of rabbit anti-mouse IgG (Jackson Laboratories, PA). Subsequently the sections were again washed in PillS(pH 7.4) and in- cubated for 30 rain with a hi00 dilution of FITC-conjugated streptavidin (Jackson Immuno Research Laboratories, Inc., Avondale, PA). Finally the sections were washed and mounted as previously described (20).

Histochemicai Staining

Serial transversesections of unfixed rabbitgracilis muscles were stained for myosin ATPase after alkalinepreincubations(pH 10.4) as described byGuth and Samaha (13). This method specifically labelsthe myosin ATPase ofonly fast skeletal muscle fibers. ImmunocoUoidai GoldLabeling Immunocolloidai gold labeling of thin sections of cryofixed, frceze-dried, and Lowicryl K4M-embedded rabbitskeletal muscle was carried out as pre- viously described (16, 17) except for the modification outlined below. The sections were first labeled with DEAE-Affi-Blue gel-purified mAb IIID5 to the ,vFsubunit of the 1,4-dihydropyridine receptor (15 ttg/ml in PBS pH 7.4). Then an afffinity-purified rabbit anti-mouse (F fragment) gamma globulin (Jackson Immuno Research Laboratories Inc.) was used at 25 ttg/ml in PBS containing 3% BSA. Finally the sections were incubated with affinity-purified goat anti-rabbit gamma globulin-colloidal gold con- jugate (3-7 nm) at 0.5 mg/ml PBS containing3% BSA 0anssen Pharmaceub ica, Beerse, Belgium). To assess the immunolabelingspecificity of the thin sections of Lowicryl K4M-embedded tissue, mouse gamma globulin purified from preimmune serum (10tzg/ml PBS) was substitutedfor rnAbIIID5 in the immunolabeling procedure. After immunolabeling, the sections were first stained for 15 s in saturated uranyl acetate in 50% ethanol (6) and then for 15 s in lead ci- trate. The sections were examined in a Hitachi 7000 transmission electron microscope. Ultrastructure of Cryofixed 7issue To assess the preservationof the ultrastructural features of the cryofixed and freeze-dried skeletal muscle tissue used for the immunoelectronmicroscop- ical studies, some of these tissues were vapor osmicated and then embedded in Spurt resin, as described by McGuffee et al. (27). Thin sections were stained with 2% uranylacetate followed by lead citrate, according to stan- dard procedures, and examined in a Hitachi 7000 transmissionelectron mi- croscope.