Immunohistochemical staining was performed with primary antibodies against ER (Ventana Medical Systems, Inc., Arizona, United States), PR (Ventana Medical Systems, Inc.), PGRMC1 (Abnova Corporation, Walnut, CA, United States. PAB20135) or PGRMC2 (Abnova Corporation. H00010424-M04). ER, PR, PGRMC1 and PGRMC2 expression was graded independently by two pathologists (Tsai HW and Ho CL) according to the percentages of stained hepatocytes or HCC cells. Because PGRMC1 is found in the cytosol and subcellular organelles[8 (link)], cytoplasmic staining was considered to be positive. High PGRMC expression was defined as more than two-thirds of the cells exhibited positive staining. In the case of ER and PR, nuclear staining was considered to be positive.
Pab20135
Manufactured by Abnova
Sourced in United States
PAB20135 is a laboratory product manufactured by Abnova. It is a piece of equipment designed for use in scientific research and analysis. The core function of this product is to facilitate specific tasks or processes within a laboratory setting. No further details about its intended use or capabilities are provided to maintain an unbiased and factual approach.
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Lab products found in correlation
4 protocols using pab20135
1
Immunohistochemical Evaluation of ER, PR, PGRMC1, and PGRMC2 in HCC
2
Immunofluorescence Analysis of Steroid Receptors
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KK-1 or HG hOEC 6 × 103 cells/well were seeded onto microscope slide coverslips and treated with vehicle (0), MF (5 μM), P4 (0.3 μM), vehicle (0), MF (3 μM), DXM (200 nM), MF (3 μM) + HSP90i (50 nM), HSP90i (50 nM) + DXM (200 nM) or vehicle (0), MF (3 μM), P4 (0.3 μM), AG-205 (1 μM), MF (3 μM) + AG-205 (1 μM), P4 (0.3 μM) + AG-205 (1 μM) in stimulation medium. KK-1 or HG hOEC cells were fixed in 3–4% PFA in PBS pH 7.4 for 15 min at room temperature and permeabilized for 20 min in 0.1% Triton X-100. To reduce autofluorescence cells were incubated with 100 mM NH4CL for 10 min. To block unspecific binding cells were incubated in blocking solution (3% BSA in PBS with 0.05% Tween 20) for 30 min. Next, cells were incubated for 1 h with primary antibodies GR (SC-56851, Santa Cruz Biotechnology; dilution 1:400), PGRMC1 (PAB20135, Abnova Corporation; dilution 1:1000) or HMGB1 (ab79823, Abcam; dilution 1:350) diluted in blocking solution. Next, cells were incubated with secondary fluorescent antibody Alexa Fluor 488 goat anti-mouse IgG (ab150113, Abcam; dilution 1:400) or Alexa Fluor 647 donkey anti-rabbit IgG (Life Technologies, dilution 1:600) for 45 min. To detect cell nuclei cells were incubated with DAPI for 1 min.
3
Dual Immunofluorescence for PGRMC1 and NENF
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To minimize autofluorescence in dual staining, tissues were treated with 100 mM of NH4Cl for 10 min. The blocking solution, a mixture of 5% NGS and 1% BSA in PBST, was then applied for 1 h at room temperature. After blocking unspecific binding sites with 3% BSA in PBS with 0.05% Tween 20 for 30 min, the tissue slides were incubated with primary antibodies for PGRMC1 (PAB20135 from Abnova Corporation, Taipei, Taiwan; dilution 1:300) and NENF (MAB6714 from R&D Systems Europe Ltd., Abingdon, UK; dilution 25 µg) diluted in the blocking solution for 1 h. Following the previous step, the tissue slides were incubated in the dark with the secondary fluorescent antibodies Alexa Fluor 594 and 488 goat anti-mouse (ab150116 from Abcam, Cambridge, UK; dilution 1:500 and ab150113 from Abcam, Cambridge, UK; dilution 1:500, respectively) for 1 h. Cell nuclei were detected by incubating the tissue slides with DAPI.
4
Immunofluorescence analysis of nuclear receptors
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BLTK-1 cells 1–2 × 104 cells/well were seeded onto microscope slide coverslips and after 16 h treated with vehicle (0), MF (5 μM, 17.5 μM), vehicle (0), MF (3 μM), DXM (200 nM), MF (3 μM) + HSP90i (50 nM), HSP90i (50 nM) + DXM (200 nM) or vehicle (0), MF (3 μM), P4 (0.3 μM), AG-205 (1 μM), MF (3 μM) + AG-205 (1 μM), and P4 (0.3 μM) + AG-205 (1 μM) in stimulation medium. Cells were fixed in 4% PFA in PBS pH 7.4 for 15 min at room temperature and permeabilized for 10 min in 0.1% Triton X-100. To reduce autofluorescence cells were incubated with 100 mM NH4CL for 10 min. After blocking unspecific binding sites with 3% BSA in PBS with 0.05% Tween 20 for 30 min cells were incubated for 1 h with primary antibodies anti-GR (SC-56851, Santa Cruz Biotechnology, Dallas, TX, USA; dilution 1:400), anti-PGRMC1 (PAB20135, Abnova Corporation; dilution 1:1000) or anti-HMGB1 (ab79823, Abcam, Cambridge, UK; dilution 1:350) diluted in blocking solution. Next, cells were incubated with secondary fluorescent antibody Alexa Fluor 488 goat anti-mouse IgG (ab150113, Abcam, Cambridge, UK; dilution 1:400) or Alexa Fluor 647 donkey anti-rabbit IgG (Life Technologies, Carlsbad, CA, USA; dilution 1:600) for 45 min. To detect cell nuclei, cells were incubated with DAPI for 1 min.
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