Harris hematoxylin

2,5-Dihydroxybenzoic acid (DHB), norharmane, Fluoroshield with DAPI, Monoclonal Anti-Actin α-Smooth Muscle antibody, LC-MS grade methanol, HPLC grade chloroform, Bouin’s solution, Triton X-100, Tween 20, poly-L-lysine solution 0.1% and Peel-a-Way embedding molds (truncated, 12 × 12 × 20 mm) were purchased from Sigma-Aldrich, Saint Louis, MO, USA. Histology-grade solvents, Eosin G or Y (alcoholic solution 0.5%) and Masson’s Trichrome staining kit were purchased from DiaPath S.p.A., Martinengo, Italy, while QA-Agarose low melting point was obtained from MP Biomedical, Santa Ana, CA, USA. Harris hematoxylin, phosphate buffer saline (PBS) tablets, bovine serum albumin (BSA), SuperFrost Plus glass slides and cover slides were purchased from VWR International, LLC, Radnor, PA, USA. Trifluoroacetic acid (TFA), Antihuman CD68 antibody and AlexaFluor 568 goat anti mouse secondary antibody were purchased from Life Technologies-Invitrogen, Carlsbad, CA, USA. LC-MS grade water was purchased from Thermo Fisher Scientific, Rockford, IL, USA, while ITO coated glass slides for MALDI were obtained from Bruker Daltonics, Billerica, MA, USA. Paraformaldehyde 4% solution was purchased from Alfa Aesar, Haverhill, MA, USA.

Hematoxylin and Eosin: Sections were incubated in Harris Hematoxylin (VWR 95057–858) for 8 min, differentiated in 1% acid alcohol for 4 s, and incubated in Bluing Reagent Solution (VWR 95057–852) for 2 min. Sections were then counterstained with Eosin-phloxine solution (VWR 95057–846). For quantification, a vacuolated neuron was defined as having 2 or more clear somal abscesses greater than 2 μm in diameter. A minimum of 200 neurons were analyzed per animal. Sudan Black B: Sections were incubated in 1% Sudan Black B (Sigma 199664-25G) in 70% ethanol for 2 min. Kohler illuminated brightfield images were acquired on an Axioskop 2 (Zeiss) with a Retiga 2000R CCD camera (QImaging). Brightness and contrast were adjusted equally between experimental conditions. Post-hoc color balance was adjusted to achieve uniform white balance on all images.

Tissues were sectioned (10 μm) using the CM1950 cryostat (Leica). For hematoxilin and eosin staining, frozen slides were first thawed and incubated in filtered Harris Hematoxylin (VWR, Radnor, PA) for 8 min before being briefly dipped in water containing 0.2% ammonium hydroxide for 50 sec. The samples were then put through a series of additional dips: 95% EtOH for 5 sec, Eosin (VWR) for 1 min, 95% EtOH for 5 sec, followed by 95% EtOH for 2 min (3X), 100% EtOH for 2 min (3X), and Xylene for 2 min (3X). Specimen were covered with Permount (Fisher Scientific, Hampton, NH). For the quantification of centrally nucleated fibers, a wheat germ agglutinin and DAPI stain was used. Briefly, slides were fixed with 4% PFA (Electron Microscopy Sciences, Hatfield, PA) for 10 min and blocked with phosphate-buffered saline solution containing 5% normal goat serum and 0.3% Triton X-100 (Sigma-Aldrich, St. Louis, MO) for 30 min. Secondary antibodies used were WGA-488 (ThermoFisher) diluted 1:500 and DAPI in blocking buffer and incubated with samples for 15 min. Slides were mounted with Fluoromount-G (Southern Biotech, Birmingham, AL). The percent of centrally nucleated fibers was manually counted with the help of the Fiji software. Kruskal-Wallis test followed by Dunn’s multiple comparisons was applied.

To clarify the effect of MSTN knockout on skeletal muscle, we performed histological analysis and statistical quantitative analyses of muscle fibers in skeletal muscle of mutant and wild-type channel catfish. Four each of one-month-old fry from mutant (individuals with frame-shift mutation) and control groups were euthanized with tricaine methane sulfonate (MS-222) (Western Chemical Inc., Ferndale, WA). Subsequently, catfish muscles were dissected, cross sectioned and fixed in 4% paraformaldehyde at room temperature for at least 24 hours, and then dehydrated and paraffin embedded using Tissue Tek II^® (Sakura Finetek USA, INC, CA). Serial sections were made at 7 μm thickness using a rotary microtome (American Optical Corporation, Southbridge, MA). The sections were mounted on glass slides and stained with the regressive staining method using Harris hematoxylin (VWR International, PA) and eosin with phloxine (Sigma-Aldrich, Inc., MO). Muscle fibers were counted. Cell numbers were calculated as the number of fibers per cross-sectional muscle area using the “Cell Counter” features of ImageJ program^{84 (link)} and used for evaluating fiber size.

Histological and statistical quantitative analyses of myofibers in breast muscles of the non-injected control group and treated groups were performed (n = 3 samples from each group, 3 sections from each sample, 9 total sections for each group). Chicken embryos on day 18 of incubation were decapitated and breast muscle tissues were sampled and preserved in 10% formalin. The samples were dehydrated and paraffin embedded. Serial sections with 6 μm thickness were produced by a rotary microtome. The sections were mounted on microscopical slides and stained with the regressive staining method using Harris hematoxylin (VWR International, LLC, Philadelphia PA, USA) and eosin with phloxine (Sigma-Aldrich, Inc., St. Louis MO, USA). Myofibers were counted and cell numbers were calculated as the fibers number per cross-sectional muscle area with the help of the “Cell Counter” features of ImageJ program. The average size of myofibers was also detected using the same features [26 (link)]. All images were obtained using bright field Leica ICC50 HD Microscope with an in-built camera (Leica Microsystems, Wetzlar, Germany).