Modified mayer s hematoxylin

Formalin-fixed lung tissues were paraffin embedded, and 5-μm-thick sections were prepared, dewaxed in xylene, and rehydrated through graded concentrations of alcohol to water. Then, antigen retrieval was performed in boiling sodium citrate buffer for 10 min. Sections were incubated with blocking buffer for 1h at room temperature, followed by overnight incubation with either anti-mouse Sema3E antibody (R&D Systems, Minneapolis, MN) or isotype control IgG (Jackson ImmunoResearch Laboratories, West Grove, Pa) at 4°C. Slides were then washed twice with TBS followed by incubation for 1h at room temperature with biotin-conjugated secondary antibody. After extensive washing with TBS, slides were incubated with streptavidin-alkaline phosphatase for 30 min at room temperature. Development was performed using Fast Red (Sigma-Aldrich, Oakville, ON, Canada) and counterstained with modified Mayer's hematoxylin (Fisher Scientific, Fair Lawn, NJ). Finally, slides were mounted and visualized using AxioVision software (Carl Zeiss, Inc, Thornwood, NY).

Three-μm formalin-fixed paraffin-embedded testis cross sections from P42 mice were deparaffinized in xylene and rehydrated to distilled water. The sections were subjected to antigen retrieval for 30 minutes at 95 °C in antigen retrieval buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0). The sections were then incubated in 0.3% hydrogen peroxide (Fisher) for 30 minutes to block endogenous peroxidase activity. Immunostaining was performed using the VECTASTAIN Elite ABC kit (Vector Laboratories) following the manufacturer’s protocol. The primary antibody, rabbit anti-cleaved caspase 3 (Cell Signaling; 9661) was diluted 1:100 and incubated with sections for 90 minutes at room temperature. Detection was performed using the ImmPACT DAB substrate (Vector Laboratories). Slides were subsequently stained with modified Mayer’s hematoxylin (Fisher), dehydrated in an ethanol gradient, coverslipped with Permount (Fisher) and imaged.

Tissues were collected and fixed in 10% neutral buffered formalin for 72 hours before embedding in paraffin wax or in OCT for frozen sections. Oil Red O staining was used to stain 5-micron frozen sections prepared using a cryostat machine. Hematoxylin and eosin (H&E) staining of 5-micron sections from formalin fixed, and paraffin embedded hepatic tissues were performed to detect alterations in morphology and structure. Tissues were heat set at 55°C for 30 minutes to prevent shredding or tissue loss and sequentially cleared through multiple changes of Citrisolv Hybrid (Decon Laboratories, Inc. King of Prussia, PA, USA), then dehydrated by sequential graded alcohol steps, staining with Modified Mayer’s Hematoxylin (Fisher Scientific, Waltham, MA, USA) and counterstained with Alcoholic Eosin Y with phloxine (Millipore Sigma, St. Louis, MO, USA). Hydrated slides were then cover slipped with permanent mounting media and imaged with an Olympus DP74 microscope and CellSens Standard v2.3 software. A second series of slides were stained with saturated picric acid containing 0.1% Sirius Red (Direct Red 80, Millipore Sigma, St. Louis, MO, USA) stain for 30 minutes to visualize collagen bundle staining (25 (link)).

Paraffinized liver was cut onto slides (4 μm) and routinely deparaffinized. Slides were microwaved (GE, Model#: JES1142WD04) on high setting in Antigen Unmasking Solution (Vector Laboratories) and cooled at room temperature. Endogenous enzyme activity was blocked with BLOXALL (Vector Laboratories) and nonspecific protein binding was blocked with 10% Normal Goat Serum (Thermo Fisher). Slides were incubated with anti-ZIKV NS2B Ab (Genetex) at 4 °C for 14 hrs. Slides were incubated with ImmPRESS HRP (Vector Laboratories) secondary Ab followed by incubation with ImmPACT NovaRED (Vector Laboratories) substrate. Slides were counterstained with Modified Mayer’s Hematoxylin (Thermo Fisher) and routinely processed for mounting. Positive, negative, and rabbit IgG (Vector Laboratories) controls were included with each batch. Morphology of immuno-reactive cells were confirmed by a board-certified pathologist, and immunoreactivity was quantified by ImageDx^TM (Reveal Biosciences).

Heart morphology was analyzed in postnatal day 0 (P0) mice and cell proliferation was analyzed by phospho-histon H3 (pHH3) staining in E12.5 hearts. Briefly, the mouse thorax was fixed in 4% paraformaldehyde overnight, dehydrated in ethanol, embedded in paraffin and serially sectioned into 5-μm sections. Heart sections were stained with hematoxylin/eosin (H/E) and images were captured using a light microscope (Observer D1, Zeiss, Germany). Images were taken on every 25 μm of the heart and the three-dimensional visualization of heart structures was reconstructed using AMIRA® program. To analyze cell proliferation and apoptosis, heart sections were immunostained using anti-pHH3 (phospho S10) antibody (Abcam) and anti-cleaved claspase-3 antibody (Cell Signaling), respectively, followed by incubation with biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA). Signals were visualized by 3-3′di-aminobenzidin tetrahydrochloride (Sigma-Aldrich Chemie, St. Louis, MO, USA). Counterstaining was performed with modified Mayer’s hematoxylin (Thermo Scientific, Waltham, MA, USA). The number of pHH3⁺ cells from at least 3 individual heart sections per sample was quantified and normalized to areas of the myocardium.

Aortic root sections were stained and quantified as previously described (Bouchareychas et al., 2015 (link)). Hearts were excised following perfusion with PBS, fixed in 10% formalin, incubated overnight in 20% sucrose and embedded in OCT. For Apoe^−/− mice, hearts were sectioned with a cryostat (10 μm), stained with oil red O (Sigma-Aldrich) and counterstained with modified Mayer’s hematoxylin (Thermo Fisher Scientific). Lesion area was quantified by averaging six sections that were spaced 50 μm apart, starting from the base of the aortic root. Necrotic core area was measured by determining the acellular region (DAPI negative) in each plaque. For macrophage staining, sections were labeled with a primary rat anti-mouse MOMA-2 antibody (Cedarlane labs) and detected with an AlexaFluor 594 donkey anti-rat IgG antibody (Thermo Fisher Scientific). To assess apoptotic cells in lesion, sections were stained for cleaved caspase-3 (Cell Signaling, 1/200) staining followed by AlexaFluor 488 donkey anti-rabbit IgG (Thermo Fisher Scientific). Total cleaved caspase 3 positive area was calculated and divided by the total atherosclerotic plaque area measured by ORO in serial sections. Images were captured with a Nikon Eclipse Ni and a Zeiss Observer microscope, analyzed using NIS-Elements and ZEN 3.0 Software. Analyses were performed in a blinded manner.