Immunohistochemical staining for ERα and PCNA was performed on all groups. Tissues sections were deparaffinized, rehydrated, and subjected to microwave antigen retrieval. Endogenous peroxidase was then blocked with 3% H2O2. Nonspecific antibody binding was blocked with 5% goat serum. Then the sections were incubated with primary antibody at 4°C overnight. Primary antibodies were respectively rabbit anti-PCNA (1:200) and rabbit anti-ERα (1:200) purchased from Abcam (Cambridge, MA, USA). The sections were incubated with the biotinylated secondary antibody for 1 h and washed in PBS. Color reactions were performed with DAB. Then the sections were counterstained with hematoxylin, dehydrated, cleared in xylene, and mounted with coverslips. Negative control sections were incubated with PBS. According to the intensity of the dye color and the number of positive cells, the scores of specimens were calculated.
Rabbit anti erα
Manufactured by Abcam
Sourced in United States
Rabbit anti-ERα is a primary antibody that specifically recognizes the estrogen receptor alpha (ERα) protein. ERα is a nuclear receptor that mediates the biological effects of the hormone estrogen. This antibody can be used to detect and study the expression and localization of ERα in various cell and tissue types.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti pcna, by Abcam (1 mentions)
Fluorchem e system, by Bio-Techne (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti pcna, by Abcam (1 mentions)
β actin, by ZSGB-BIO (1 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid protein assay, by Keygen Biotech (1 mentions)
Odyssey system, by LI COR (1 mentions)
Mouse β actin, by Cell Signaling Technology (1 mentions)
Rabbit anti erβ, by Abcam (1 mentions)
Rabbit anti caspase 3, by Cell Signaling Technology (1 mentions)
Rabbit anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Rabbit anti bcl 2, by Cell Signaling Technology (1 mentions)
Rabbit anti ucp1, by Abcam (1 mentions)
Dmi8 inverted microscope, by Leica (1 mentions)
Ix70 inverted microscope, by Olympus (1 mentions)
Nile red, by Merck Group (1 mentions)
Alexa fluor 488 conjugated donkey anti rabbit igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Rabbit anti map2, by Santa Cruz Biotechnology (1 mentions)
6 protocols using rabbit anti erα
1
Immunohistochemical Analysis of ER-α and PCNA
2
Western Blot Analysis of ERα and PCNA
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Western blotting analysis for ERα and PCNA was performed on all groups. The total protein was extracted, mixed with 5 × SDS-PAGE sample buffer at a 4:1 ratio, and denatured by heating the sample to 99°C for 10 min. After electrophoresis on SDS-PAGE, the proteins were separated. Then the target proteins were transferred to signed polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). After incubation with 5% skim milk, the membranes were incubated with primary antibody for 18 h at 4°C. The primary antibodies for target proteins were respectively rabbit anti-ERα (1:150) and anti-PCNA (1:200) obtained from Abcam. Then the membranes were incubated with horseradish peroxidase conjugated secondary antibody for 1 h at room temperature with agitation. Bands were detected using the FluorChem E system (Bio-Techne, San Jose, CA, USA). The data were expressed as the relative net intensity of the target protein/β-actin (ZSBIO, Beijing, China). At least 3 independent biological replicates were performed.
3
Western Blot Analysis of Protein Expression
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Whole cell lysates were prepared with radio immunoprecipitation assay lysis buffer. Total proteins were isolated and quantified by bicinchoninic acid protein assay (KeyGen Biotech, China). The proteins were boiled in the loading buffer, electrophoresed on 10% sodium dodecyl sulfate–polyacrylamide gel, transferred onto nitrocellulose membranes, and blocked in 5% nonfat dry milk. Afterward, the cells were incubated with primary antibody (mouse anti-ClC-3, Abcam; rabbit anti-ERα, Abcam) and then with horseradish peroxidase-labeled secondary antibody (goat anti-mouse IgG, goat anti-rabbit IgG; Invitrogen). The target proteins were detected by chemiluminescence detection method.
4
Estrogen Receptor Signaling in Ischemic Stroke
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We measured the protein levels of ERα and ERβ, and levels of phospho‐ERα‐Ser118 in the cortex and striatum of non‐ischemic mice; and measured Bcl‐2 and caspase‐3 protein expression in the ischemia penumbra, which were dissected as follows: in the coronal section of the brain, a sagittal section was made 2 mm apart from the middle suture, then the innermost uninjured boundary point was found, and a section from this point to the front sagittal section was made, and the area between the two sections is the ischemic penumbra (Wang et al., 2009 (link)). The ischemic penumbra in the first antibody include: rabbit anti‐ERα (1:500, Abcam), rabbit anti‐ERβ (1:500, Abcam), rabbit anti‐phospho‐ERα‐Ser118 (1:500, Abcam), rabbit anti‐Bcl‐2 (1:1000, Cell Signaling Technology), rabbit anti‐cleaved‐caspase‐3 (1:1000, Cell Signaling Technology), rabbit anti‐caspase‐3 (1:1000, Cell Signaling Technology), or mouse β‐actin (1:1000, Cell Signaling Technology) antibodies. Protein bands were visualized using a LI‐COR Odyssey System (LI‐COR Biotechnology) and were densitometrically analyzed by automated ImageJ software (NIHImage, Version 1.61).
5
Immunohistochemical Analysis of Adipose Tissues
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Tissues were formalin-fixed, paraffin-embedded, sectioned and stained with haematoxylin and eosin (H&E) as described.21 (link) Immunohistochemistry and immunofluorescence were done as described.20 (link),21 (link) Following de-paraffinization, the immunostaining procedure of paraffin-embedded tissue sections was similar to that of frozen tissues and cultured cells. Primary antibodies: rabbit anti-UCP1 (1:200, Abcam, Cambridge, MA), rabbit anti-ERα (1:200, Abcam), rabbit anti-mouse β3-adrenergic-receptor (1:200, Abcam), mouse anti-PCNA (1:200, Millipore, Burlington, MA) and goat-anti-Perilipin (1:200, BD Biosciences, San Jose, CA). Secondary antibodies: Goat/Donkey anti- rabbit/mouse conjugated with AlexaFluor-488/Cy3, and Donkey anti-goat conjugated with Cy5 (1:500, Jackson ImmunoResearch, West Grove, PA). DAPI (1:500, Fluka, Milwaukee, WI). Nile-red (1 µg/mL, Sigma). Bright-field and fluorescent images were collected on: Olympus inverted IX70 microscope, Olympus upright BX40 microscope, Leica inverted DMi8 microscope or Leica upright DM6 microscope. An independent researcher reviewed slides to confirm results.
6
Immunofluorescence Staining of Neuronal Markers
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Treated cells were fixed with ice-cold 100% methanol at -20°C for 15 min and washed 3 times with phosphate buffered saline (PBS) for 5 min. Non-specific binding was blocked by incubating cells in 5% BSA blocking solution (containing 10% horse serum in 1X TBS-T) for 60 min at room temperature. This was followed by washing with PBS. Thereafter, the cells were incubated with the following primary antibodies overnight at 4°C: rabbit anti-NF-B p65 (Santa Cruz; 1:100), rabbit anti-ERα (Abcam; 1:100), rabbit anti-ERβ (Santa Cruz; 1:100) and rabbit anti-MAP2 (Santa Cruz; 1:100). Following overnight incubation, cells were washed thrice with PBS and incubated for 2 h in the dark with Alexa Fluor 488conjugated donkey anti-rabbit IgG secondary antibody (Life Technologies; 1:500).
Thereafter, cells were washed with PBS and counterstained with DAPI for 5 min. After rinsing cells with PBS, excess buffer was removed and gold antifade reagent (Invitrogen) was added. All staining procedures were performed at room temperature. Fluorescence images were obtained using EVOS® FLoid® cell imaging station.
Thereafter, cells were washed with PBS and counterstained with DAPI for 5 min. After rinsing cells with PBS, excess buffer was removed and gold antifade reagent (Invitrogen) was added. All staining procedures were performed at room temperature. Fluorescence images were obtained using EVOS® FLoid® cell imaging station.
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