Paraffin-embedded sections of the mouse aorta were taken, dewaxed, dehydrated, and subjected to antigen retrieval. Sections were added with 0.03% Triton for 10 min and sealed with normal goat serum blocking solution (C-0005, HaoranBio, Shanghai, China) for 60 min at room temperature. For macrophages, macrophages were fixed with 4% paraformaldehyde for 30 min, washed with precooled PBS And sealed as the above. Next, primary antibodies of HMGB1 (66525-1-Ig, 1:250, Mouse, Proteintech, VA, USA), F4/80 (ab6640, 1:50, Rabbit, Abcam), GSDMD (sc-393581, 1:200, Mouse, SANTA, UC, USA), iNOS (ab210823, 1: 100, Mouse, Abcam), and CD206 (60143-1-Ig, 1:200, Mouse, Proteintech) were selected for sample incubation at 4 ℃ overnight. Then, the samples were incubated with fluorescent secondary antibody, Alexa Fluor® 647-Anti-Rabbit IgG (ab150075, 1:500, Donkey, Abcam) or Alexa Fluor® 488-Anti-Mouse IgG (ab150113, 1:500, Goat, Abcam) at room temperature for 60 min in the dark. A fluorescence microscope was utilized for observation, with fluorescence intensity recorded.
Alexa fluor 488 anti mouse igg
Manufactured by Abcam
Sourced in United Kingdom, Japan
Alexa Fluor-488 anti-mouse IgG is a secondary antibody conjugated with the Alexa Fluor-488 fluorescent dye. It is designed to detect and bind to mouse immunoglobulin G (IgG) antibodies in various immunoassays and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 647 anti rabbit igg, by Abcam (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Ab210823, by Abcam (1 mentions)
Ab150075, by Abcam (1 mentions)
Ab150113, by Abcam (1 mentions)
Ab6640, by Abcam (1 mentions)
Sc 393581, by Abcam (1 mentions)
Iba1 antibody, by Fujifilm (1 mentions)
Anti cd68, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg cy5, by Abcam (1 mentions)
Dapi fluoromount g, by Southern Biotech (1 mentions)
Tcs sp8, by Leica (1 mentions)
Ab7788, by Abcam (1 mentions)
Cm1950, by Leica (1 mentions)
Sm22α, by Abcam (1 mentions)
Alexa fluor 647 anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Abcam (1 mentions)
Ve cadherin, by Merck Group (1 mentions)
Alexa fluor 488 anti mouse igg, by Thermo Fisher Scientific (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
8 protocols using alexa fluor 488 anti mouse igg
1
Immunofluorescent Staining of Mouse Aorta
2
Multimarker Immunohistochemistry of Brain Slices
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Slices were rinsed in 0.3% Triton X-100/PBS (3 × 5 min). In order to prevent non-specific binding, slices were pre-incubated with blocking buffer (1% bovine serum albumin (BSA) and 0.3% Triton X-100 in PBS) for 2 h at room temperature. Slices were incubated with Iba1 antibody (WAKO, 019-19741; 1:1000), mouse Ab-T1 (0.5 μg/ml), or anti CD68 (Santa Cruz Biotechnology, sc-20060 AF488; 1:200) overnight at 4 °C. Slides were rinsed with 0.3% Triton X-100 in PBS (3 × 5 min) followed by incubation with Cy5 goat anti-Rabbit IgG (Abcam, ab6564; 1:200) or Alexa Fluor 488 anti-Mouse IgG (Abcam, ab150113; 1:200) in blocking buffer for additional 2 h at room temperature. Slides were washed 3 times in 0.3% Triton X-100/PBS and a drop of mounting buffer (DAPI Fluoromount-G. Cat#: 0100-20, SouthernBiotech) was added before visualized on a confocal laser scanning microscope (TCS SP8, Leica).
3
Immunofluorescence Staining of LL37 and H. pylori
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O.C.T compound embedded tissues were sectioned with a thickness 10 µm (Leica CM1950), washed in PBS, and post‐fixed in methanol. The samples were then blocked in 5% goat serum/3% BSA/0.1% Triton X‐100/PBS blocking solution, followed by incubation with primary anti‐LL37 (mouse, Santa Cruz sc‐166770) or anti‐H. pylori (rabbit, Abcam ab7788) antibodies at 4 °C for overnight. Alexa Fluor™ 488 anti‐mouse IgG (Abcam) or Alexa Fluor™ 647 anti‐rabbit IgG (Abcam) were used as secondary antibodies, respectively. Hoechst 33342 (Life Technologies) was used to stain cell nucleus. Sections were evaluated by confocal microscopy (Leica SP8).
4
Immunofluorescence Staining of Cell Lines
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HSC-4 cells cultured on cover glass (Matsunami, #5001) were washed with PBS, fixed with ice-cold acetone/methanol (1:1), blocked with 2% FBS for 40 min, and incubated with primary antibodies to E-cadherin (Cell Signaling Technology, #14472S) and vimentin (Abcam, #ab2547). The molecules were then visualized with secondary antibodies: Alexa Fluor-488 anti-mouse IgG (Invitrogen, #A21202) and Alexa Fluor-647 anti-rabbit IgG (Invitrogen, #A31573). Nuclei were stained with Hoechst 33342 (Invitrogen, #H1399). HUAEC monolayers cultured on collagen type I-coated cover glasses (IWAKI, #11-0071) were washed with PBS, fixed with 4% paraformaldehyde solution for 15 min, and permeabilized with 0.1% Triton X-100 for 15 min. The samples were then blocked with 2% FBS for 40 min and incubated with anti-VE-cadherin (Sigma-Aldrich, #MABT129) and SM22α (Abcam, #GR3321006-1) primary antibodies, followed by incubation with secondary antibodies: Alexa Fluor-488 anti-mouse IgG and Alexa Fluor-647 anti-rabbit IgG. Hoechst 33342 was used to visualize nuclei. Stained HSC-4 cells or HUAECs were mounted and observed with a Keyence BZ-X710 fluorescence microscope or a Leica TCS SP8 confocal laser scanning microscope. Quantitative analysis of obtained images (five fields of view from at least four independent samples) was done with Fiji (ImageJ).
5
Immunofluorescence Analysis of CD68 and CD163 in Breast Cancer
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The formalin-fixed, paraffin-embedded biopsy specimens of BC and benign breast disease were obtained from Shanghai Jiao Tong University Affiliated Sixth People's Hospital. Sections of 5 µm thickness were cut from the blocks and mounted on silanized slides, which were then deparaffinized and subjected to antigen retrieval for double immunofluorescent (IF) staining as previously described. 19, 20 The specimens were incubated in blocking solution overnight at 4°C with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). The blocking solution was aspirated, and mouse anti-CD68 (Abcam, UK) primary antibodies were added in blocking solution for 1-2 h at room temperature. The slides were rinsed three times with PBS and then incubated with Alexa Fluor-488 anti-mouse IgG (Abcam) for 1 h at room temperature. After the slides were washed with PBS, rabbit anti-CD163 (Abcam) was added in blocking solution for an additional 4 h, and the slides were subsequently washed. Alexa Fluor-647 anti-rabbit IgG (Abcam) was then added to each slide and incubated for 30 min at room temperature. As a negative control, no primary antibody was added. IF images were acquired using a Confocal Laser Scanning Microscope (Nikon, Japan) to identify the double-positive region of interest.
6
Visualizing RIP1 in IL-1β-Treated Chondrocytes
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Rat chondrocytes were treated with or without 10 ng/mL IL-1β for 24 h, rinsed in PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. Triton X-100 was used to penetrate the cell membrane for 5 min, and goat serum was applied to block non-specific binding sites. Then the cells were incubated with primary antibodies against RIP1 (BD Biosciences, 610459; 1:200) at 4°C overnight, washed with PBS and incubated with anti-mouse IgG (Alexa Fluor® 488) (Abcam, ab150113; 1:1,000). Nuclei were counterstained with Hoechst 33342 for 10 min. After washed again, the samples were visualized using confocal microscope.
7
Immunofluorescence Assay for X. laevis MyoD
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Immunofluorescence was carried out on whole X. laevis embryos overexpressing with HA-tagged MyoD proteins according to Christen and Slack, 1999, [25 (link)]. Embryos fixed in MEMFA and stored in methanol at −20 °C were gradually rehydrated and then permeabilised in potassium dichromate treatment followed by 5% H2O2 in PBS. After washes and an hour blocking at room temperature, embryos were incubated overnight at 4 °C in BBT (PBS + 1%BSA, 0.1% Triton X-100) and 5% horse serum with a 1:1000 concentration anti-HA antibody (Sigma). The secondary anti-mouse IgG Alexa Fluor 488 (Abcam, Cambridge, UK) was used at 1:1000 and embryos were incubated for 1 h at room temperature.
8
HER2 Expression Quantification in Breast Cancer Cells
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Cultured breast cancer cells were fixed in 10% formalin, and the samples (2 × 106 cells) were mixed with PBS containing 5% fetal bovine serum, 1 mM EDTA, and 0.1% NaN3. The samples were immunostained with 5 μg/mL primary antibody to HER2 (Anti-C-ErbB2/c-Neu(Ab-5) Mouse mAb(TA-1), Calbiochem, Merck Millipore, Tokyo, Japan) at 4 °C for 30 min. After a PBS wash, the samples were incubated with 1 μg/mL secondary antibody (Anti-Mouse IgG-Alexa Fluor488, Abcam, Tokyo, Japan) at 4 °C for 30 min. After a PBS wash, the samples were mixed with dedicated buffer for FACS and subjected to FACS measurements (MACSQuant Analyzer, Miltenyi Biotec, Bergisch Gladbach, Germany). We measured the fluorescence intensity per cell of 20,000 cells by FACS19 (link) and calculated the mean value. Fluorescence-labeled beads (QIFIKIT) were also measured for calibration. QIFIKIT is used to determine the density of antibody-binding antigen per cell using FACS.
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