For immunohistochemistry, the primary antibodies used were rabbit anti-cleaved caspase 3 (1:1000; Cat#9661, Cell Signaling Technology, Beverly, MA), mouse anti-Ki67 (1:200; Cat#556003, BD Pharmingen, Heidelberg, Germany), rat anti-BrdU (1:200; Cat#OBT0030G, Accurate Chemical), mouse anti-NeuN (1:500; Cat#MAB377, Chemicon). The secondary antibodies used were Alexa Fluor 555-conjugated goat anti-mouse IgG (1:1000; Cat#A21422, Molecular Probes), Alexa Fluor 488-conjugated goat anti-rat IgG (1:1000; Cat#A21208, Molecular Probe), Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:1000; Cat#4412, Cell Signaling), Alexa Fluor 555-conjugated donkey anti-goat IgG (1:1000; Cat#A21432, Molecular Probes), Alexa Fluor 647-conjugated goat anti-mouse IgG (1:1000; Cat#A21237, Molecular Probes). For western blots, the primary antibodies were, rabbit anti-p-aPKCζ/ι (T410/403)(1:500, Cat#9378, Cell Signaling), mouse anti- aPKCζ/ι (1:500; Cat#610175, BD), rabbit anti-p-CREB (S133)(1:500; Cat#9198, Cell Signaling), mouse anti- CREB (1:500; Cat#9104, Cell Signaling), rabbit anti-p300 (1:100; Cat#sc-585, Santa-Cruz), and mouse anti-p300 (1:1000, Cat#ab14984, Abcam). Secondary antibodies for western blots were HRP-conjugated goat anti-mouse or anti-rabbit (1:4000; Cat#7076 and #7074, Boehringer Mannheim).
Alexa fluor 488 conjugated goat anti rabbit igg
Manufactured by Cell Signaling Technology
Sourced in United States
Alexa Fluor 488-conjugated goat anti-rabbit IgG is a secondary antibody that binds to rabbit primary antibodies. It is labeled with the fluorescent dye Alexa Fluor 488, which can be detected using fluorescence-based techniques.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 594 conjugated goat anti mouse igg, by Cell Signaling Technology (3 mentions)
Rabbit anti iba1, by Abcam (2 mentions)
Alexa fluor 594 conjugated goat anti rabbit igg, by Cell Signaling Technology (2 mentions)
Normal goat serum (ngs), by Merck Group (2 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 555 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated goat anti rat igg, by Thermo Fisher Scientific (1 mentions)
Rabbit anti p creb s133, by Cell Signaling Technology (1 mentions)
Rabbit anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Mouse anti neun, by Merck Group (1 mentions)
Alexa fluor 555 conjugated donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Mouse anti creb, by Cell Signaling Technology (1 mentions)
Rabbit anti p300, by Santa Cruz Biotechnology (1 mentions)
Mouse anti ki67, by BD (1 mentions)
Ab180606, by Abcam (1 mentions)
Ab6123, by Abcam (1 mentions)
Sc 376845, by Santa Cruz Biotechnology (1 mentions)
Sc 166412, by Santa Cruz Biotechnology (1 mentions)
16 protocols using alexa fluor 488 conjugated goat anti rabbit igg
1
Immunohistochemistry and Western Blot Protocols
2
Colocalization of β5i and RIG-I
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To explore the colocalisation of β5i and RIG-I, double-label immunostaining was performed. After blocking with 5% bovine serum albumin in PBST at room temperature for 1 hour, the samples were treated with mouse anti-RIG-I monoclonal antibody (1:50, sc-376845, Santa Cruz Biotechnology) and rabbit anti-β5i monoclonal antibody (1:2000, ab180606, Abcam) overnight at 4°C, followed by three washes with PBS (10 min each wash). Next, the samples were treated with Alexa Fluor 594-conjugated goat anti-mouse IgG (1:200, 8890, Cell Signaling Technology) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200, 4412, Cell Signaling Technology), which were used as the secondary antibodies, in a dark chamber for 1 hour, followed by three washes with PBST. After counterstaining with DAPI (Beyotime, Shanghai, China), the slides were mounted with coverslips. To clarify whether β5i is expressed in regenerative myocytes, we performed double-label immunostaining using rabbit anti-β5i monoclonal antibody (1:2000, ab180606, Abcam) and mouse anti-NCAM1 monoclonal antibody (1:500, ab6123, Abcam), following the same procedure mentioned above. In addition, double-label immunofluorescence staining using rabbit anti-β5i monoclonal antibody and mouse anti-MxA monoclonal antibody (1:100, sc-166412, Santa Cruz) was performed following the above procedure.
3
Multimodal Immunolabeling of Brain Slices
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Immunofluorescence was performed as described previously [22 (link)]. After blocking with 10% normal goat serum (Sigma-Aldrich, USA), slices were incubated with primary antibodies as following: rabbit anti-GFAP (1:300; Cell Signaling Technology, USA), rabbit anti-IBA1 (1:400; Abcam, Cambridge, MA, USA), rabbit anti-beta Amyloid (1:50; Abcam, Cambridge, MA, USA), and mouse anti-Olig1 (1:100; Santa Cruz, USA). After incubated overnight at 4 °C and washed three times in 0.01 M PBS (3 × 5 min), slices were incubated with the following secondary antibodies for 1 h at room temperature: Alexa Fluor® 594 conjugated goat anti-rabbit IgG or Alexa Fluor® 488 conjugated goat anti-rabbit IgG or Alexa Fluor® 488 conjugated goat anti-mouse IgG(1:1000; Cell Signaling Technology). Slices were mounted in ProLong® Gold antifade reagent (Thermo Fisher Scientific, USA) prior to imaging.
4
Immunofluorescent Analysis of DNA Damage
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143B-GFP-Luc Cells (1 × 105 per well) were plated into 6-well plates with coverslips on the bottom and then treated with vehicle, doxorubicin (40 nM), sorafenib (4 μM) and miR-34a (12 nM) alone or in combination on the next day. 24 hours later, cells were fixed with 10% formaldehyde for 15 min at room temperature, rinsed three times in PBS, and blocked for 1 h in PBS consisting of 2% bovine serum albumin/0.3% Triton X-100 (Fisher Scientific) at room temperature. After rinsed three times in PBS, cells were incubated with anti-γH2A.X (Ser139) antibody (1:800, Cell Signaling Technology, Danvers, MA) overnight at 4 °C. After washed with PBS for 3 times, cells were incubated in Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1:800, Cell Signaling Technology) for 1 h at room temperature in dark. Nucleus was counterstained with DAPI using Prolong® Gold Antifade Reagent (Cell Signaling Technology). Images were taken with a Zeiss Axio Observer.z1 Microscope coupled to a Zeiss LSM 710 Scanning Device (Zeiss, Oberkochen, Germany).
5
Immunohistochemical Analysis of BDNF and S100 in Sciatic Nerve
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Frozen sections of sciatic nerve tissue or RSC96, which had been fixed at 48 h after transfection were blocked with 10% goat serum and incubated with anti-BDNF (1:100; Abcam) or S100 (1:200; Proteintech, Chicago, United States) at 4°C overnight. After washing with PBS, the samples were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (Cell Signaling Technology, Beverly, United States) for 1 h at room temperature and counterstained with Hoechst 33342 (Biyuntian Biotechnology, Shanghai, China) or 4′,6-diamidino-2-phenylindole (DAPI). The immunostained samples were observed and photographed under a fluorescence microscope (IX53, Olympus, Japan).
6
Cardiac Fibrosis Quantification via Immunofluorescence
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Murine hearts were fixed in 4% formaldehyde, embedded in paraffin and cut into 4‐µm sections. Masson trichrome staining of heart tissue sections was used to detect cardiac fibrosis. For immunofluorescence, sections were deparaffinized and rehydrated, and antigen retrieval was performed using citrate buffer, pH 6 (Sigma‐Aldrich). In brief, sections were permeabilized and blocked in 5% goat serum and 0.3% TritonX‐100 in PBS and incubated with polyclonal antibodies against α‐SMA (1:1000, Abcam) overnight at 4°C. Then, sections were washed in PBS and incubated with Alexa Fluor 488‐conjugated goat anti‐rabbit IgG (Cell Signaling) followed by incubation with DAPI. Fluorescence images were acquired using a Nikon Eclipse Ti‐U 100 inverted microscope.
7
Immunofluorescence Analysis of Hep3b Cells
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When Hep3b cells were cultured to the appropriate densities, cells in co-culture plates were fixed with 4% paraformaldehyde for 10 min at room temperature and subsequently permeabilized with 0.1% Triton X-100 for 10 min. Following blocking with 5% BSA and 10% horse serum (Sigma-aldrich; Merck KGaA) in PBST for 1 h at room temperature, Hep3b cells were incubated with primary antibody against Ki-67 (1:200; cat. no. #12075; Cell Signaling Technology, Inc.) overnight at 4˚C. Following the primary incubation, cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200; cat. no. ab150077; Abcam) for 1 h at room temperature. Cell nuclei were stained with DAPI for 5 min at room temperature. Hep3b cells were washed three times with PBST prior to observation under a light microscope at x200 magnification and images were captured (Olympus Corporation).
8
Quantification of DNA Damage Foci
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Cells grown on coverslips were fixed with 3% paraformaldehyde at room temperature for 20 min and then permeabilized with PBS containing 0.5% Triton X-100 for 5 min. The coverslips were blocked with PBS containing 5% goat serum for 30 min, and immunostained with anti-γH2AX (Millipore) antibody at 4°C overnight, then washed for at least three times and incubated with secondary antibody, Alexa Fluor 488 conjugated goat anti-rabbit IgG (1:50, Cell Signaling, US), for 1 hour at room temperature. DAPI (4, 6-diamidino-2-phenylindole, Sangon biotech, Shanghai, China) was used to counterstain the nuclei. Photographs were captured by Olympus Laser scanning confocal microscopy (Olympus optical Co., Tokyo, Honshu, Japan). At least 50 cells were scored for each treatment group, and the number of γH2AX foci was counted.
9
Quantifying Leukocytes in Rat Lung Tissue
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Frozen lung tissue samples of rats were sectioned at 7 µm intervals and fixed with 4% paraformaldehyde. For the detection of MPO-positive cells (neutrophils, monocytes), sections were permeabilized with 0.2% Triton X-100 and incubated with 5% normal goat serum + 0.5% BSA + 0.2% TritonX-100 in PBS in order to block unspecific binding. Sections were further incubated with anti-MPO antibody (1:100, Abcam) for 1 h followed by additional incubation with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:1000, Cell Signaling Technology) overnight at 4 °C.
For the detection of leukocytes, a mouse monoclonal anti-rat CD45 antibody was used (clone OX-1, 1:20, BD Pharmingen). Then, slides were further incubated with Alexa Fluor 555-conjugated goat anti-mouse IgG (H + L) secondary antibody (1:1000, Thermo Fisher) for 1 h at room temperature. Nuclei were counterstained with 4′,6-diamidino-2-phenyl indole (DAPI) and slides were mounted in fluorescence mounting medium (Dako). Tissue sections were examined using an inverted microscope (Eclipse Ti-U 100, Nikon). Signal intensity of CD45 immunolabeling in ten random fields was quantified as mean value and averaged using the Image J analysis software.
For the detection of leukocytes, a mouse monoclonal anti-rat CD45 antibody was used (clone OX-1, 1:20, BD Pharmingen). Then, slides were further incubated with Alexa Fluor 555-conjugated goat anti-mouse IgG (H + L) secondary antibody (1:1000, Thermo Fisher) for 1 h at room temperature. Nuclei were counterstained with 4′,6-diamidino-2-phenyl indole (DAPI) and slides were mounted in fluorescence mounting medium (Dako). Tissue sections were examined using an inverted microscope (Eclipse Ti-U 100, Nikon). Signal intensity of CD45 immunolabeling in ten random fields was quantified as mean value and averaged using the Image J analysis software.
10
Immunofluorescence Staining of IGF2BP3
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PCa cells were grown on coverslips (Corning, USA), and mixed with 4% paraformaldehyde for 30 min. Then the cells were permeabilized with 0.1% TritonX-100. Subsequently, the cells were incubated with Tris-buffered saline containing 5% bovine serum albumin (BSA) for 30 min. Afterwards, samples were incubated with antibodies specific for IGF2BP3 (Abcam, USA, 1:100) at 4 °C overnight. Finally, coverslips were treated with the fluorescent secondary antibody Alexa Fluor 488-conjugated goat anti-rabbit IgG (#4412S, Cell Signaling Technology) (1250 dilution) and DAPI (300 nmol/L) staining. The images were photographed under a Nikon AISi Laser Scanning Confocal Microscope (Nikon instruments Inc., Japan).
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