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Tissue tek

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Tissue-Tek is a tissue processing system designed for the preparation of tissue samples for microscopic examination. It is used to dehydrate, clear, and impregnate tissue samples with paraffin wax, enabling the creation of thin sections for histological analysis.

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Lab products found in correlation

Cm1860, by Leica camera (2 mentions) Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions) Cas block histochemical reagent, by Thermo Fisher Scientific (2 mentions) Asma cy3, by Merck Group (2 mentions) Isolectin b4 af568, by Thermo Fisher Scientific (2 mentions) Vectashield plus antifade mounting medium with dapi, by Vector Laboratories (2 mentions) Superfrost plus slide, by Thermo Fisher Scientific (1 mentions) Vectashield mounting medium, by Vector Laboratories (1 mentions) Cryostat, by Leica Biosystems (1 mentions) Formaldehyde, by Electron Microscopy Sciences (1 mentions) Penicillin and streptomycin, by Thermo Fisher Scientific (1 mentions) Prolong gold antifade reagent with 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions) Alexa conjugated phalloidin, by Thermo Fisher Scientific (1 mentions) Image it fx signal enhancer, by Thermo Fisher Scientific (1 mentions) Pcr reagents, by Promega (1 mentions) Supersignal west femto chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Cytotox 96, by Promega (1 mentions) Non fat dry milk, by Bio-Rad (1 mentions) Protein determination reagent, by Bio-Rad (1 mentions) Axioscope 2, by Zeiss (1 mentions)

11 protocols using tissue tek

1

Immunofluorescent Characterization of Excised NGP Tumors

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Excised NGP tumors were embedded in Tissue-Tek® optimum cutting temperature (O.C.T.) compound (Electron Microscopy Services), then stored at -20 ºC until cryosectioned (Leica CM1860). 15 μm thick cryosections were fixed with acetone and permeabilized with Tween 20. After blocking non-specific binding with CAS-Block Histochemical Reagent (ThermoFisher Scientific), the following primary antibodies were used: murine iNOS (1:500, #13120, Cell Signaling), aSMA-Cy3 (1:1000, #C6198, Sigma), and pimonidazole (1:100, #Pab2627, Omnikit, Hypoxyprobe, Massachusetts). Isolectin-B4-AF568 (1:100, #I21412, Invitrogen) was diluted in HEPES buffer. Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) secondary antibody was applied following incubation in primary solution and a series of washing steps. Finally, the slides were mounted with DAPI (VECTASHIELD PLUS Antifade Mounting Medium with DAPI, Vector Laboratories).
Bellary A., Nowak C., Iwanicki I., Flores-Guzman F., Wu L., Kandel J.J., Laetsch T.W., Bleris L., Hernandez S.L, & Sirsi S.R. (2023). Non-viral nitric oxide-based gene therapy improves perfusion and liposomal doxorubicin sonopermeation in neuroblastoma models. Theranostics, 13(10), 3402-3418.
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2

Cryosectioning and Immunostaining of Retinal Tissue

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Eyeballs were fixed in 4% paraformaldehyde for 10 or 60 minutes (for Gβ3 staining), rinsed in phosphate buffer, soaked overnight at 4°C in 0.1M phosphate buffer containing 30% sucrose for cryoprotection and embedded in a mixture of two parts 20% sucrose in phosphate buffer and one part optimal cutting temperature (OCT) compound (Tissue Tek, Electron Microscopy Sciences, Hatfield, PA, USA). Radial sections (15–18 μm) were cut on a cryostat (Leica Biosystems, Buffalo Grove, IL, USA) at −20°C, and sections were collected on a superfrost plus slide (Fisher scientific, Pittsburgh, PA, USA). Sections were permeabilized and blocked with 10% normal goat serum, 5% sucrose and 0.5% Triton X-100 in phosphate buffer for 1 hour at 20°C. Sections were then incubated in primary antibodies (diluted in blocking solution) at 4°C overnight or 3 days for mGluR6. Retinas were washed 3 times in 0.1M phosphate buffer containing 5% sucrose at room temperature. Sections were then incubated in secondary antibodies at 20°C for 3 hours, washed, mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) and cover slipped. For double labeling, sections were simultaneously incubated in both primary antibodies (raised in different hosts) followed by both secondary antibodies.
Tummala S.R., Dhingra A., Fina M.E., Li J.J., Ramakrishnan H, & Vardi N. (2016). Lack of mGluR6-Related Cascade Elements leads to Retrograde Trans-synaptic Effects on Rod Photoreceptor Synapses via Matrix-associated Proteins. The European journal of neuroscience, 43(11), 1509-1522.
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3

Amylase Assay and Cell Culture Reagent Protocol

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Phadebas Amylase Assay kit was purchased from Magle Life Sciences. Cholecystokinin (CCK-8) was purchased from Research Plus. Dulbecco’s minimal essential medium (DMEM), essential amino acids, fetal bovine serum (FBS), TrypLE Express, penicillin and streptomycin, Alexa-conjugated Phalloidin, Image-iT® FX Signal Enhancer and Prolong gold antifade reagent with 4,6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen. Bovine serum albumin, 12-O-tetradecanoylphorbol-13-acetate (TPA) and a protease inhibitor cocktail containing AEBSF, aprotinin, EDTA, leupeptin, and E64 were purchased from Calbiochem. Protein determination reagent and non-fat dry milk were purchased from Bio-Rad Life Science Research. DNA Maxi, Mini-prep kits, PCR reagents, and CytoTox 96 were purchased from Promega. The QuikChange XL Site-Directed Mutagenesis Kit, restriction enzymes were purchased from Fermentas. Trypsin substrate Boc-Glu-Ala-Arg-MCA, and cathepsin B substrate Arg-Arg-MCA were purchased from Peptides International. Formaldehyde and Tissue Tek were purchased from Electron Microscopy Sciences. SuperSignal West Femto Chemiluminescent Substrate was purchased from Thermo Scientific. YM201636 was purchased from Chemdea. Apilimod was purchased LGM Pharma.
Messenger S.W., Thomas D.D., Cooley M.M., Jones E.K., Falkowski M.A., August B.K., Fernandez L.A., Gorelick F.S, & Groblewski G.E. (2015). Early to Late Endosome Trafficking Controls Secretion and Zymogen Activation in Rodent and Human Pancreatic Acinar Cells. Cellular and Molecular Gastroenterology and Hepatology, 1(6), 695-709.
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4

Immunofluorescence Analysis of Retinal Proteins

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The cornea and lens were removed and the remaining eyecup fixed with 4% formaldehyde in PBS for 15 min on ice. The tissues were rinsed 3 times in PBS, placed in 30% sucrose overnight at 4°C, embedded in OCT (Tissue Tek, Electron Microscopy Science), and 10 μm frozen sections were obtained. Tissue sections were blocked for 1 h in PBS containing 1% horse serum and 0.1% Triton X-100. Sections were then incubated with antibody against ARR1 (1:200 dilution) (Chen et al., 2006 (link); Rose et al., 2017 (link)) and rhodopsin (1D4, 1:1000) (MacKenzie et al., 1984 (link)), followed by 1 h incubation with a secondary anti-mouse antibody (#TI-2000, Vector Laboratories). Images were obtained by Zeiss Axioscope 2 using the same exposure time for each antibody.
Hsieh C.L., Yao Y., Gurevich V.V, & Chen J. (2022). Arrestin Facilitates Rhodopsin Dephosphorylation in Vivo. The Journal of Neuroscience, 42(17), 3537-3545.
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5

Immunostaining Protocol for Retinal Sections

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Immunostaining was carried out as previously described (Pan et al., 2014 (link); Kerov et al., 2018 (link)). Briefly, eyes (for animals less than postnatal day 8) or posterior eyecups were collected by dissection, fixed in 4% paraformaldehyde at room temperature for 15–45 min, cryoprotected in 30% sucrose, and then frozen in O.C.T. (Tissue-Tek, Electron Microscopy Sciences, Hatfield, PA). Radial sections were cut and collected on electrostatically charged glass slides, and either labeled immediately or stored at −80°C until use. Blocking buffer consisted of 10% normal goat serum and 0.5% Triton X-100 in PBS. Primary and secondary antibodies (diluted in blocking buffer) were incubated on retinal sections for 1–3 h at room temperature or overnight at 4°C. Images were acquired on a THUNDER Imager (Leica DM6B microscope equipped with a Leica DFC9000 GT camera). Computational clearing of z-stacks was performed using LASx software.
Laird J.G., Kopel A., Lankford C.K, & Baker S.A. (2023). Mouse all-cone retina models of Cav1.4 synaptopathy. Frontiers in Molecular Neuroscience, 16, 1155955.
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6

Visualizing Coronary Arteriole Proteins

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Coronary arterioles were embedded in OCT compound (Tissue-Tek; Electron Microscopy Sciences, Hatfield, PA, USA) and frozen sections (10 μm thickness) were fixed in 4% paraformaldehyde for immunohistochemical analysis as described previously [83 (link),84 (link)]. Immunolabelling was performed using a mouse monoclonal antibody against eNOS (610297, 1:100 dilution; BD Biosciences, Franklin Lakes, NJ, USA) and a rabbit polyclonal antibody against PKCβ2 (sc-210, 1:100 dilution; Santa Cruz Biotechnology, Dallas, TX, USA). The slides were then incubated with rhodamine red-labeled (Jackson Laboratories, West Grove, PA, USA) and FITC-labeled (Jackson Laboratories) secondary antibodies. Staining control tissues were exposed for the same duration to non-immune serum (Jackson Laboratories) in place of primary antibody. Slides were observed for red (rhodamine red for PKCβ2) and green (FITC for eNOS) images under a fluorescence microscope (Axiovert 200, Zeiss, Jena, Germany) and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA) as described previously [20 (link)].
Thengchaisri N., Hein T.W., Ren Y, & Kuo L. (2021). Activation of Coronary Arteriolar PKCβ2 Impairs Endothelial NO-Mediated Vasodilation: Role of JNK/Rho Kinase Signaling and Xanthine Oxidase Activation. International Journal of Molecular Sciences, 22(18), 9763.
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7

Muscle Fiber Typing and Characterization

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Samples of the CMM from each recording site were collected immediately after recording, flash-frozen with liquid nitrogen and stored at −80 °C. Soleus muscles were obtained and prepared in a similar manner. Tissues were then immersed in TissueTek (Electron Microscopy Sciences) and cryosectioned at a thickness of 20 µm before both H&E and fiber type staining protocols were performed. Standard H&E staining protocol was followed (Thermo Fisher, Halethorpe, MD). For fiber typing, samples were permeabilized in a solution of 0.3% Triton × (Sigma-Aldrich) in 1× PBS, blocked with a Mouse on Mouse blocking reagent (Vector Labs, Burlingame, CA), and then labeled with antibodies to identify type I myosin heavy chain (A4.840, Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA) and type II myosin heavy chain (My32, Thermo Fisher).
Tallon C., Russell K.A., Sakhalkar S., Andrapallayal N, & Farah M.H. (2015). Length-dependent axo-terminal degeneration at the neuromuscular synapses of type II muscle in SOD1 mice. Neuroscience, 312, 179-189.
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8

Immunostaining of Retinal Cryosections

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All antibodies used in this study are listed in Table 2.32 (link),51 (link) Immunostaining was carried out as previously described.44 (link) Briefly, posterior eyecups were collected by dissection, fixed in 4% paraformaldehyde at room temperature for 15 to 20 minutes, cryoprotected in 30% sucrose, and then frozen in OCT (Tissue-Tek; Electron Microscopy Sciences, Hatfield, PA, USA). Radial sections were cut and collected on electrostatically charged glass slides and either labeled immediately or stored at −80°C until use. Blocking buffer consisted of 10% normal goat serum and 0.5% Triton X-100 in PBS. Primary and secondary antibodies (diluted in blocking buffer) were incubated on retinal sections for 1 to 3 hours at room temperature or overnight at 4°C. Images were collected with a 63×, numerical aperture 1.4, oil-immersion objective on either a Zeiss LSM710 confocal (Carl Zeiss, Oberkochen, Germany) or an Olympus FluoView 1000 microscope (Olympus Corp., Tokyo, Japan).
Laird J.G., Gardner S.H., Kopel A.J., Kerov V., Lee A, & Baker S.A. (2019). Rescue of Rod Synapses by Induction of Cav Alpha 1F in the Mature Cav1.4 Knock-Out Mouse Retina. Investigative Ophthalmology & Visual Science, 60(8), 3150-3161.
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9

Immunohistochemical Analysis of Mouse Retina

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Antibodies and lectins used in this study are listed in Table 1. Mouse eyes from at least 3 individual mice were enucleated and the posterior eyecups collected by dissection. Eyecups were fixed in 4% paraformaldehyde at room temperature for 60–90 min, cryoprotected in 30% sucrose, and then frozen in O.C.T (Tissue-Tek, Electron Microscopy Sciences, Hatfield, PA, USA) before collecting 12 μm cryosections. For immunostaining, the sections were permeabilized in PBS containing 0.5% Triton X-100 for 10 min, followed by incubation in 10% goat serum to block nonspecific labeling. Primary antibodies were incubated on sections overnight at 4 °C, washed and secondary antibodies along with WGA and Hoechst were added for 1–2 hours at room temperature. Fluoromount-G was used as the mounting media and No 1.5 cover slips were sealed in place with nail polish. Images were collected using a 20x objective, with digital zoom set to capture the span of the retina from the OS to the GC and at 1 μm steps in the z-axis, with the pinhole set to 1 AU on a Zeiss 710 confocal microscope (Central Microscopy Research Facility, Univ. Iowa). Maximum through stack projections were made using Zen Light 2009 (Carl Zeiss); manipulation of images was limited to rotation, cropping, and adjusting the brightness and contrast levels using Zen Light 2009 or Photoshop CC (Adobe).
Inamdar S.M., Lankford C.K., Laird J.G., Novbatova G., Tatro N., Whitmore S.S., Scheetz T.E, & Baker S.A. (2018). Analysis of 14-3-3 isoforms expressed in photoreceptors. Experimental eye research, 170, 108-116.
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10

Immunofluorescent Characterization of Excised NGP Tumors

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Excised NGP tumors were embedded in Tissue-Tek® optimum cutting temperature (O.C.T.) compound (Electron Microscopy Services), then stored at -20 ºC until cryosectioned (Leica CM1860). 15 μm thick cryosections were fixed with acetone and permeabilized with Tween 20. After blocking non-specific binding with CAS-Block Histochemical Reagent (ThermoFisher Scientific), the following primary antibodies were used: murine iNOS (1:500, #13120, Cell Signaling), aSMA-Cy3 (1:1000, #C6198, Sigma), and pimonidazole (1:100, #Pab2627, Omnikit, Hypoxyprobe, Massachusetts). Isolectin-B4-AF568 (1:100, #I21412, Invitrogen) was diluted in HEPES buffer. Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) secondary antibody was applied following incubation in primary solution and a series of washing steps. Finally, the slides were mounted with DAPI (VECTASHIELD PLUS Antifade Mounting Medium with DAPI, Vector Laboratories).
Bellary A., Nowak C., Iwanicki I., Flores-Guzman F., Wu L., Kandel J.J., Laetsch T.W., Bleris L., Hernandez S.L, & Sirsi S.R. (2023). Non-viral nitric oxide-based gene therapy improves perfusion and liposomal doxorubicin sonopermeation in neuroblastoma models. Theranostics, 13(10), 3402-3418.
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