Frozen sections of cancerous and noncancerous liver tissues from HCC patients were prepared in optimal cutting temperature compound (Ted Pella Inc., Redding, CA, USA), placed on glass slides, thawed, washed with phosphate-buffered saline (PBS), and fixed with 1% paraformaldehyde for 10 min in PBS. After blocking for 1 h with PBS containing 1% bovine serum albumin and 1 mM EDTA, the slides were washed with PBS and incubated overnight at 4 °C with the primary monoclonal Abs (5 μg/mL). Next, the slides were washed with PBS thrice and incubated for 30 min with 1:1000 Alexa 488-conjugated goat anti-mouse immunoglobulin G (IgG) (Fab′)2 fragment or Alexa 568-conjugated goat anti-rabbit IgG (Fab′)2 fragment (Molecular Probes, Eugene, OR, USA) in PBS containing 0.05% Tween-20. Subsequently, the slides were washed thrice with PBS and cover-slipped using Vector-shield (Vector Laboratories Inc., Burlingame, CA, USA) containing 10-μg/mL of Topro-3 (Molecular Probes). The slides were observed using conventional fluorescent or confocal microscopy (510 meta; Zeiss, Oberkochen, Germany).
Optimal cutting temperature compound
Manufactured by Ted Pella
Sourced in United States
Optimal Cutting Temperature (OCT) compound is a water-soluble, inert embedding medium used to support and protect specimens during cryosectioning. It is designed to maintain the structural integrity of the sample while enabling thin, uniform sections to be cut.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Prolong diamond antifade, by Thermo Fisher Scientific (2 mentions)
Pna fluorescein, by Vector Laboratories (2 mentions)
Horse anti mouse igg fluorescein, by Vector Laboratories (2 mentions)
Axiovert 200 microscopy system, by Zeiss (2 mentions)
Apotome imaging, by Zeiss (2 mentions)
Pe anti mouse cd19 antibody, by Thermo Fisher Scientific (2 mentions)
Axiovision software, by Zeiss (2 mentions)
To pro 3, by Thermo Fisher Scientific (1 mentions)
510 meta, by Zeiss (1 mentions)
Vectorshield, by Vector Laboratories (1 mentions)
Axioplan 2 microscope, by Zeiss (1 mentions)
Af2644, by R&D Systems (1 mentions)
Hoechst 33342 dye, by Merck Group (1 mentions)
Ab6586, by Abcam (1 mentions)
Cy3 conjugated anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions)
Vectashield with 4 6 diamidino 2 phenylindole, by Vector Laboratories (1 mentions)
5 protocols using optimal cutting temperature compound
1
Immunofluorescent Staining of Liver Tissues
2
Detailed Immunofluorescence Kidney and Spleen Analysis
3
Quantitative Immunofluorescence Imaging of Kidney and Spleen
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Kidneys frozen in liquid nitrogen were embedded in Optimal Cutting Temperature compound (Ted Pella Inc.). Tissue was cryosectioned (5 μm) and permeabilized with cold acetone for 20 min at room temperature. The slides were washed with PBS and blocked with 2% horse serum prepared in 3% BSA/PBS for 20 min at room temperature. The sections were stained for 30 min with 1:200 diluted horse anti‐mouse IgG‐Fluorescein (Vector Laboratories) followed by extensive washing in PBS containing 0.1% Tween 20 and mounting using ProLong Diamond Antifade (Thermo Fisher Scientific) containing DAPI counterstain. Images were acquired using Axiovert 200 microscopy system with Apotome imaging and Axiovision software (Carl Zeiss Microscopy, LLC). For GC studies, spleen sections were rehydrated for 30 min with PBS and blocked for 1 h with 10% BSA/PBS at room temperature. The sections were stained for 1 h with 1:100 diluted PNA‐Fluorescein (Vector Laboratories) or anti‐GL7‐Alexafluor 488 and washed three times with PBS, followed by incubation with 1:50 diluted PE‐anti‐mouse CD19 antibody (Thermo Fisher Scientific) at room temperature for 1 h. The slides were washed and mounted using ProLong Diamond Antifade (Thermo Fisher Scientific) containing DAPI. Germinal centers were mapped using the selection tool and quantified using ImageJ (NIH). H & E stained spleen sections were also imaged for GC analyses.
4
Immunostaining of Ovarian Markers
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Ovarian tissues were fixed in 4% paraformaldehyde and either embedded in paraffin for routine histological (hematoxylin and eosin [H&E]) and immunofluorescent (IF) analyses or frozen in optimal cutting temperature compound (Ted Pella, Inc.) for other specific IF analyses. Primary antibodies specific for GAS1 (AF2644; R&D system), PTX3 (P0496; Sigma Aldrich), SFRP4 (HPA009712; Sigma Aldrich), PECAM-1 (553373; BD Pharmingen), and collagen IV (ab6586; Abcam) were used in the IF studies. Cell nuclei were counterstained with 5μg/ml Hoechst 33342 dye (Sigma Aldrich). Digital images of H&E and IF staining were captured using a Zeiss AxioPlan2 microscope in the Integrated Microscopy Core, and images of IF staining were captured with Zeiss LSM780 confocal microscope in the Optical Imaging and Vital Microscopy Core at Baylor College of Medicine.
5
Immunofluorescence Staining of Cx26 in Cryosections
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Skin was fixed in a 1% formaldehyde in PBS for 1 h at room temperature. Tissues were rinsed with PBS, immersed in Optimal Cutting Temperature compound (Ted Pella, Redding, CA), and frozen. 8–10 μm sections were cut on a cryotome, dried onto glass slides, and stained with polyclonal rabbit antibodies against Cx26 (Zymed, San Francisco, CA), washed with 0.1% Triton X-100/PBS, incubated with Cy3-conjugated anti-rabbit secondary antibodies (Jackson ImmunoResearch, West Grove, PA), washed with 0.1% Triton X-100/PBS, and mounted using Vectashield with 4′,6-diamidino-2-phenylindole (Vector, Burlingame, CA).
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