Histogel

Manufactured by StatLab

HistoGel is a tissue embedding medium designed for use in histological procedures. It is a clear, water-soluble gel that provides a stable matrix to support and orient tissue samples during sectioning and subsequent staining or analysis.

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5 protocols using histogel

Immunofluorescence Staining of Cardiomyocytes

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All cells were fixed directly on the membranes with 4% paraformaldehyde (PFA). The mESC-CMs were further processed for staining as described previously (Hinderer et al., 2015 (link)). For hESC-CMs samples, HistoGel (American MasterTech) was dispensed on the samples after PFA was removed, followed by embedding in paraffin. IF staining of 3 μm sections was performed as described previously (Votteler et al., 2013 (link)). All antibodies are listed in the Supplemental Information. DAPI was used to visualize cell nuclei. An LSM710 confocal microscope (Carl Zeiss) was used for imaging. Sarcomere length was analyzed using ImageJ (version 1.46r, Wayne Rasband, NIH) (Wang et al., 2013 (link), Nance et al., 2015 (link)).

Shen N., Knopf A., Westendorf C., Kraushaar U., Riedl J., Bauer H., Pöschel S., Layland S.L., Holeiter M., Knolle S., Brauchle E., Nsair A., Hinderer S, & Schenke-Layland K. (2017). Steps toward Maturation of Embryonic Stem Cell-Derived Cardiomyocytes by Defined Physical Signals. Stem Cell Reports, 9(1), 122-135.

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Cytological Specimen Preparation Protocol

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Direct smears on glass slides were either air-dried and Diff-Quick stained to allow immediate verification of the adequacy and quality of the specimen or fixed in 95% ethanol for ulterior Papanicolaou stain. Material for molecular analysis was obtained by rinsing the needle with saline into a tube after sample smears were done together with the material obtained from an additional pass. Any residual clot or tissue in the hub of needles was removed carefully and kept in the same tube. Cell blocks were obtained by centrifugation of the tube, then the concentrated material was supported in HistoGel™ (American Master Tech, CA. United States) following the manufacturer’s instructions. The cell button was processed as a conventional biopsy and embedded in paraffin.

Pedrosa L., Araujo I.K., Cuatrecasas M., Soy G., López S., Maurel J., Sánchez-Montes C., Montironi C., Saurí T., Sendino O., Pérez F.M., Ausania F., Fernández-Esparrach G., Espósito F.M., Vaquero E.C, & Ginès A. (2023). Targeted transcriptomic analysis of pancreatic adenocarcinoma in EUS-FNA samples by NanoString technology. Frontiers in Molecular Biosciences, 10, 1161893.

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Histological Analysis of Bypass Graft Tissue

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Upon retrieval of surgical samples, the lumen of the bypass segment was flushed with cold saline, filled with prewarmed Histogel (American MasterTech, Lodi, CA) and fixed in 10% neutral buffered formalin overnight then stored in 70% ethanol until further histologic processing. Specimens were embedded in paraffin and sectioned at 3 μm thickness. Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) were performed per standard protocol [10 (link),12 (link),14 (link)]. Staining was done using antibodies against αSMA (Dako, Carpinteria, CA and Abcam, Cambridge, MA), GFP (Aves Labs, Tigard, OR), CDX2 (Dako), E-cadherin (Dako), lysozyme (Abcam), chromogranin A (Immunostar, Hudson, Wisconsin), synaptophysin (Dako), Muc2 (Santa Cruz), and CD10 (Dako). For immunofluorescent stains, appropriate secondary antibodies conjugated to Alexa Fluor 488 or 594 (Life Technologies, Carlsbad, CA) were added at 1:200 dilution and nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI, Life Technologies).

Khalil H.A., Hong S.N., Rouch J.D., Scott A., Cho Y., Wang J., Lewis M.S., Martin M.G., Dunn J.C, & Stelzner M.G. (2019). Intestinal epithelial replacement by transplantation of cultured murine and human cells into the small intestine. PLoS ONE, 14(5), e0216326.

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Histological Characterization of Intestinal Tissue

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Intestinal tissue was fixed overnight in 10% neutral buffered formalin and stored in 70% ethanol until further histologic processing. Intestinal cultures were fixed in 10% buffered formalin for 5 minutes then resuspended in 30 μL preheated Histogel (American MasterTech) and stored in ethanol as above. Specimens were embedded in paraffin and sectioned at 3 μm thickness. Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) were performed per standard protocol (Lahar et al. 2011 (link); Jabaji et al. 2013 (link)). Staining was done using antibodies against CD10 (56C6, Dako, Carpinteria, CA), lysozyme (ab74666, Abcam, Cambridge, MA), chromogranin A (20086, Immunostar, Hudson, WI) (Gonzalez et al. 2013 (link)), villin (1D2C3, Dako), E-cadherin (NCH-38, Dako), β-catenin (β-Catenin-1, Dako), p120-catenin (98, Ventana, Tucson, AZ), and cytokeratin 8 and 18 (EP17/EP30, Dako). Goblet cells were identified with the periodic acid-Schiff (PAS) stain (Brown et al. 1988 (link)). Porcine ISEMFs were plated in 0.95 cm² culture wells, fixed as above, and stained with antibodies against α-smooth muscle actin (α-SMA, M0851, Dako), desmin (M0760, Dako), and vimentin (ab92547, Abcam). For immunofluorescent stains, conjugated goat secondary antibody (Invitrogen) was added at 1:200 dilution and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen).

Khalil H.A., Lei N.Y., Brinkley G., Scott A., Wang J., Kar U.K., Jabaji Z.B., Lewis M., Martín M.G., Dunn J.C, & Stelzner M.G. (2016). A Novel Culture System for Adult Porcine Intestinal Crypts. Cell and tissue research, 365(1), 123-134.

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Fine-Needle Aspiration Cell Preparation

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The needle of each fine-needle aspiration pass after the creation of smears was rinsed in BD CytoRich red preservative fluid or Hanks balanced salt solution. The cells in these solutions were centrifuged, and the pellet was mixed gently with a few drops of molten HistoGel (American Master Tech, Lodi, Calif) at a temperature of approximately 508C. The HistoGel and the cell pellet were allowed to solidify and then were poured into a nylon-mesh biopsy bag and fixed in formalin for 2 to 12 hours, and then they were embedded in paraffin as a routine histology block.

Harada S., Agosto-Arroyo E., Levesque J.A., Alston E., Janowski K.M., Coshatt G.M, & Eltoum I.A. (2015). Poor cell block adequacy rate for molecular testing improved with the addition of Diff-Quik-stained smears: Need for better cell block processing. Cancer cytopathology, 123(8).

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