Immunohistochemistry (IHC) was performed as published previously [47 (link)] [7 (link)]. Tumor tissues were dissected, fixed in 4% formalin overnight and processed in paraffin wax. Signal detection was performed semiautomatically in the Autostainer 480 S (Medac, Hamburg, Germany). Nuclei were stained by hematoxylin. Immunofluorescence staining (IF) was performed as published [15 (link)] [47 (link)]. Nuclei were counterstained by Hoechst 33342. See S1 Table for antibody details and dilution ratios.
Autostainer 480 s
Manufactured by Medac
Sourced in Germany
The Autostainer 480 S is a compact, automated slide staining instrument. It is designed to perform routine staining procedures on microscope slides. The Autostainer 480 S can handle a variety of staining protocols and is suitable for use in clinical laboratories and research settings.
Automatically generated - may contain errors
Lab products found in correlation
Bright vision polymer detection system, by Medac (3 mentions)
Dab 415192f, by Medac (2 mentions)
Ab58610, by Abcam (2 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Ber h2, by Agilent Technologies (1 mentions)
P mtor, by Cell Signaling Technology (1 mentions)
Dab detection kit, by Roche (1 mentions)
Ventana benchmark ultra, by Roche (1 mentions)
Ventana benchmark ultra autostainer, by Roche (1 mentions)
Bx50 microscope, by Olympus (1 mentions)
Bond 3 ihc stainer, by Leica (1 mentions)
C dpvb 500 hrp, by Medac (1 mentions)
15 protocols using autostainer 480 s
1
Immunohistochemistry and Immunofluorescence Protocols
2
Immunohistochemical Analysis of PRM1 and PRM2
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Bouin’s fixed testicular tissue was processed in paraffin wax. Immunohistochemistry (IHC) against PRM1 was performed as described previously64 (link) using the Autostainer 480 S (Medac, Hamburg, Germany). For IHC of PRM2, sections were treated for 10 min at RT with decondensing-mix containing 25 mM dithiothreitol, 0.2% Triton X-100 and 200 IU heparin/ml in PBS to enhance antigen accessibility for the primary antibody69 (link). Subsequently, sections were washed in 0.02 M PBS (pH 7.4), boiled for 20 min in sodium citrate buffer, treated for 20 min with 3% H2O2 in methanol and blocked for 20 min with 5% bovine serum albumin (BSA) in PBS. Incubation with the primary antibody was performed overnight at 4 °C, with biotinylated goat-anti-mouse secondary antibody (DAKO) and the Vectastain ABC Kit (Vector Laboratories, Burlingame, CA, USA) for 1 h at RT each. Immunoreaction was visualized using AEC (DAKO). Finally, sections were counterstained with hematoxylin and covered with glycerin gelatin.
3
Immunohistochemical Analysis of TCam-2 Cells
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Immunohistochemistry (IHC) was performed as published previously [10 (link)]. Tumor tissues were dissected, fixed in 4 % formalin overnight and processed in paraffin wax. Signal detection was performed semiautomatically in the Autostainer 480 S (Medac, Hamburg, Germany). Nuclei were stained by hematoxylin. See Table S1 for antibody details and dilution ratios. For IHC, 5 tumor tissues from TCam-2-ΔSOX2 clones and 1 tumor tissue from 2102EP cells as control was analyzed.
4
Immunohistochemical Analysis of Tumor Tissues
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Immunohistochemistry was performed as published [8 (link)]. Briefly, tumor tissues were dissected, fixed and processed in paraffin wax. Tissue sections on glass slides were pre-treated in the Lab Vision PT Modul (Thermo Scientific, Munich, Germany) and in PT Modul Buffer (pH 6) (TA-250-PM, Medac, Hamburg, Germany). Endogenous peroxidases were blocked by incubation in peroxidase blocking buffer (TA-125-HP, Medac) for 10 min. Primary antibodies were incubated for 30 min at room temperature. Signal detection was performed semiautomatically in the Autostainer 480 S (Medac) using the Bright Vision + polymer detection system (Medac) and the following settings: Enhancer for 10 min, polymer for 20 min, DAB (415192F, Medac) for 8 min. Afterwards, nuclei were stained by hematoxylin for 3 min. See Table 2 for antibody details and dilution ratios.
5
Immunohistochemistry Staining and Quantification
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Immunohistochemistry was performed as described previously 20 , 25 . Tumours were dissected, fixed in 4% formalin at 4°C overnight and processed in paraffin wax. Staining was performed semiautomatically in the Autostainer 480 S (Medac, Hamburg, Germany). For antibody details, see Table 1 . Stainings were quantified from three individual tumours (n = 3). Significance was calculated using two‐tailed Student's t‐test.
6
Immunohistochemistry and Immunofluorescence Protocols
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IHC was performed as published previously (Nettersheim et
al, 2015 ). Tumour tissues were dissected, fixed in 4%
formalin overnight and processed in paraffin wax. Signal detection was performed
semi-automatically in the Autostainer 480S (Medac, Hamburg, Germany). Nuclei were
stained by hematoxylin. IF was performed as described previously (Nettersheim et al, 2011c (link), 2015 (link)). Nuclei were stained by Hoechst 33342. SeeTable 1 for antibody details and dilution ratios.
al, 2015
formalin overnight and processed in paraffin wax. Signal detection was performed
semi-automatically in the Autostainer 480S (Medac, Hamburg, Germany). Nuclei were
stained by hematoxylin. IF was performed as described previously (Nettersheim et al, 2011c (link), 2015 (link)). Nuclei were stained by Hoechst 33342. See
7
Immunohistochemical Detection of 8-OHdG
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Dissected tissues were fixed in Bouin’s solution at 4°C overnight and processed in paraffin wax. Sections of 3 µm thickness were obtained on glass slides, deparaffinized, rehydrated and treated with decondensing buffer (25 mM DTT, 0.2% (v/v) Triton x-100 and 200 i.U./mL heparin in PBS) for 1 min at 37°C to increase antigen accessibility. Sections were further processed in the Lab Vision PT module (Thermo Fisher Scientific) and Autostainer 480S (Medac, Hamburg, Germany) as published previously [40 (link)]. The primary antibody against 8-OHdG (clone 15A3, Santa Cruz Biotechnology, Dallas, TX, USA) was used at a dilution of 1:200.
8
Immunohistochemical Analysis of CD30 Expression
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A total of 4 × 104 tumour cells in PBS/1.5% BSA were cytospun at 12000 g for 5′ onto glass slides and air‐dried for 15′. Signal detection was performed semiautomatically in the Autostainer 480 S (Medac, Wedel, Germany) using the Bright Vision+ polymer detection system (Medac) and the following settings: anti‐CD30 primary antibody (BER‐H2, dilution 1:200, Dako, Eching, Germany) for 20′, enhancer for 10′, polymer (Poly‐HRP‐Goat anti‐mouse/‐rabbit‐IgG) for 20′, 3,3′‐diaminobenzidine (DAB) (415192F, Medac) for 8′. Nuclei were stained by haematoxylin for 3′.
9
Subcutaneous Xenograft Tumor Assay
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A total of 5 × 106 TSCs were resuspended in 200 μl CM containing FGF4 and were injected subcutaneously into male nude mice. After 7 days, lesions were dissected, fixed in 4% paraformaldehyde overnight, embedded in paraffin, and sectioned (4 μm). Sections were stained with hematoxylin and eosin or subjected to routine immunohistochemical staining protocols, followed by incubation with primary α-Tfap2c antibody (1:500; 6E4/4; Santa Cruz Biotechnology) or α-CD31 (1:25; Dianova) for 30 min at room temperature. Signal detection was performed semiautomatically in the Autostainer 480 S (Medac) using the Bright Vision+ polymer detection system (Medac).
10
Immunohistochemical Analysis of Spermatogenesis
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Bouin's fixed 5-μm thick sections were processed for immunohistochemistry. Staining was carried out using a Lab Vision PT module (Thermo Fisher Scientific) and Autostainer 480S (medac) as described previously (Umer et al., 2021 (link)). Primary antibodies against HOOK1 (1:200; 10871-1-AP) (Zhou et al., 2009 (link)), LC3B (1:100; ab58610, Abcam), ARL3 (1:200; 10961-1-AP) (Qi et al., 2013 (link)), GOPC (1:200; 12163-1-AP, Thermo Fisher Scientific), COPA (1:200; BS-12464R, Thermo Fisher Scientific), PICK1 (1:200; PA5-76084, Thermo Fisher Scientific), SP56 (1:200; MA1-10866, Thermo Fisher Scientific), acrosin (1:200; BS-5151R, Thermo Fisher Scientific), AKT1 (1:100; 4060, Cell Signaling Technology), SQSTM1 (1:200; 5114, Cell Signaling Technology) and ARF3 (1:200; 10800-1-AP, Proteintech) were used.
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