Brain coronal sections were fixed with 4% paraformaldehyde for 30 min and then permeabilized with 0.3% Triton X-100 (Amresco, Washington, USA) for 30 min at room temperature. Later on, they were blocked with 5% bovine serum albumin (BSA, Amresco) solution for 1 h at 37 °C. They were then incubated with anti-NeuN (1:250, HUABIO, Hangzhou, Zhejiang, China), anti-Iba-1 (1:500, Wako, Tokyo, Japan) or anti-GFAP (1:200, HUABIO) antibodies overnight at 4 °C. After washing in PBS, they were incubated with secondary antibodies of Alexa Fluor 555 labeled donkey anti-mouse (1:1000, Thermo Fisher) or Alexa Fluor 488 labelled goat anti-rabbit IgG (1:500, Thermo Fisher) for 1 h at 37 °C, respectively. Nuclei were stained with DAPI (BOSTER, Wuhan, Hebei, China) for 1 min. Pictures were subsequently taken on a fluorescence microscope.
Alexafluor 488 labelled goat anti rabbit igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
AlexaFluor 488-labelled goat anti-rabbit IgG is a secondary antibody conjugated with the fluorescent dye AlexaFluor 488. It is used to detect and visualize primary rabbit antibodies in immunoassays and microscopy applications.
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Lab products found in correlation
Anti phospho histone h3 ser10 antibody, by Abcam (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Boster Bio (1 mentions)
Triton x 100, by Avantor (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
Tcs sp5, by Leica (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Tbs tween, by Merck Group (1 mentions)
Tween 20, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Goat serum, by Merck Group (1 mentions)
Digital imaging system, by Nikon (1 mentions)
Alexa fluor 488 labelled goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Inverted fluorescence microscope, by Olympus (1 mentions)
Arrayscan vti hcs instrument, by Thermo Fisher Scientific (1 mentions)
Thymidine, by Merck Group (1 mentions)
Arrayscan vti hcs, by Thermo Fisher Scientific (1 mentions)
Ab1566, by Merck Group (1 mentions)
Lsm 880 confocal laser scanning microscope, by Zeiss (1 mentions)
8 protocols using alexafluor 488 labelled goat anti rabbit igg
1
Fluorescent Labeling of Brain Cell Types
2
Immunofluorescent Staining of Endothelial Tight Junctions
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After exposure to shear, HUVEC were fixed with 4% PFA for 10 min, permeabilised with 0.1% Triton X-100 (Sigma-Aldrich, UK) for 5 min and blocked with 1% bovine serum albumin (BSA) for 1 h at room temperature. Cells were then incubated with rabbit anti-human zonula occludens-1 (ZO-1) (#13663, Cell Signaling Technology, USA) at a 1:200 dilution in 1% BSA overnight at 4 °C, followed by three washes in PBS and incubation with Alexa Fluor 488-labelled goat anti-rabbit IgG (A11008, ThermoFisher Scientific, USA) at a 1:300 dilution in PBS at room temperature for 1 h. Nuclei were stained with DRAQ5 as above. Secondary antibody was imaged with the inverted Leica TCS SP5 using a 10× objective, 488 nm excitation and detection at 505-535 nm. DRAQ5 was imaged using the wavelengths described above.
3
Immunofluorescence Staining of Paraffin-Embedded Sections
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When gels were recovered from mice, they were processed as previously described for paraffin embedding33 (link). Briefly, gels were fixed in 4% neutral buffered formalin overnight and subsequently dehydrated and paraffin embedded. Sections of 4 µm were cut and processed for either hematoxylin eosin staining or for immunofluorescence staining.
After dewaxing, sections were placed in Tris–HCl buffered saline supplemented with Tween-20 (TBS-Tween, 0.1 M Tris HCl, 0.14 M NaCl, 0.05% Tween-20, pH7.6, all reagents from Sigma-Aldrich) for 10 min, then incubated at room temperature for 1 h in TBS-Tween supplemented with 10% goat serum (Sigma-Aldrich) and then for 1 h at room temperature with the primary antibody. Sections were then washed 3 times with TBS-Tween, the secondary antibody applied and incubated for another hour. Sections were then counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) and then mounted.
As primary antibody, human cytokeratin 14 (CK14, 1:500 dilution, polyclonal AF-64, Covance, Princeton, NJ, USA) was used, while as secondary antibody, AlexaFluor 488-labelled goat anti-rabbit IgG was used (1:500, Invitrogen, Thermo Fisher Scientific).
After dewaxing, sections were placed in Tris–HCl buffered saline supplemented with Tween-20 (TBS-Tween, 0.1 M Tris HCl, 0.14 M NaCl, 0.05% Tween-20, pH7.6, all reagents from Sigma-Aldrich) for 10 min, then incubated at room temperature for 1 h in TBS-Tween supplemented with 10% goat serum (Sigma-Aldrich) and then for 1 h at room temperature with the primary antibody. Sections were then washed 3 times with TBS-Tween, the secondary antibody applied and incubated for another hour. Sections were then counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) and then mounted.
As primary antibody, human cytokeratin 14 (CK14, 1:500 dilution, polyclonal AF-64, Covance, Princeton, NJ, USA) was used, while as secondary antibody, AlexaFluor 488-labelled goat anti-rabbit IgG was used (1:500, Invitrogen, Thermo Fisher Scientific).
4
Immunofluorescence Staining of Baculovirus-Expressed IgT Protein
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Sf9 III cells infected with Bac-IgT virus were preserved with 4% paraformaldehyde (PFA) three days post infection. After fixation, the cells were permeabilized with 0.1% Triton X-100. The expressed IgT protein was stained with 1:200 dilution of mouse or rabbit anti-CH2-CH4 IgT antibodies at room temperature for 1 hour. The cells were then incubated with 1:1000 dilution of Alexa fluor 488 labelled goat anti-mouse IgG (Invitrogen, Gaithersburg, USA) or Alexa fluor 488 labelled goat anti-rabbit IgG (Invitrogen, Gaithersburg, USA) and counterstained with DAPI (Invitrogen). The fluorescence signal was detected with an inverted fluorescence microscope (Olympus, UK) and the images were captured by a digital imaging system (Nikon, Japan).
5
HeLa Cell Cycle Synchronization and Mitosis Quantification
6
High-Content Screening of Mitotic Cells
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Cells were plated at 10 000/well in 96-well plates and incubated overnight. The following day compound stocks in DMSO were diluted in medium then added to cells with a maximum final DMSO concentration on cells of 0.2%. Cells were incubated with compound for 24 h then fixed in 3.7% formaldeyde. Cells were permeabilised with 0.1% Triton X-100 then incubated with anti-phospho-histone H3 (Ser10) antibody (Abcam ab5176). The cells were washed with PBS then incubated with AlexaFluor 488 labelled goat anti-rabbit IgG (Invitrogen A11034) in the presence of 4ug/ml Hoechst 33342 (Invitrogen H3570). Cells were washed in PBS then imaged on an Arrayscan VTi HCS instrument (Thermo Fisher) using the Target Activation V4 Bioapplication. A user-defined threshold was applied to identify mitotic cells based on the intensity of phospho-histone H3 staining.
7
Substance P Expression in DRG Neurons
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L4 DRGs ipsilateral to the cancer-bearing limbs were surgically extracted, fixed in 4% paraformaldehyde, and cryostat sectioned at 25 µm thick transverse to the longitudinal axis. The sections were washed in PBS and incubated with blocking solution containing 10% normal goat serum at 25℃ for 2 h. After overnight incubation with 1:500 rabbit anti-substance P (Millipore, AB1566, Billerica, MA) at 4℃, sections were washed in PBS and incubated with AlexaFluor 488 labelled goat anti-rabbit IgG (Invitrogen, Canada) diluted 1:2000 at 25℃ for 2 h and viewed using a fluorescent microscope. For DRGs containing neurobiotin-injected neurons, these were also incubated with NeutrAvidin Texas Red 1:100 (Vector Laboratories) at 25℃ for 2 h to reveal neurobiotin staining via light microscopy.
8
Visualizing DNA Damage Response in HELQ-Deficient Cells
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WT U2OS-DR and HELQ−/− U2OS-DR cells were seeded onto four-chamber tissue culture slides (Millipore) and treated the next day with 4 µM camptothecin or 10 Gy irradiation. For siRNA knockdown, cells were transfected with non-targeting or HELQ siRNA, incubated overnight and seeded onto chamber slides the next day. Cells were treated with the designated damaging agent 48 h after siRNA transfection. At the designated time points, cells were fixed, blocked and permeabilized. Cells were stained with the following antibodies: anti-phosphorylated-histone H2A.X (Ser139) (05-636, Millipore Sigma-Aldrich), anti-RAD51 (PC130 Millipore Sigma-Aldrich), anti-RPA32 (2208, Cell Signaling Technology), anti-phosphorylated-RPA32 (S4/S8) (ab87277, Abcam). Secondary antibodies were as follows: AlexaFluor 488-labelled goat anti-rabbit IgG, AlexaFluor 568-labelled donkey anti-mouse IgG, AlexaFluor 568-labelled goat anti-rat (Invitrogen). Images were obtained using a Zeiss LSM 880 confocal laser scanning microscope and analysed using ImageJ. At least 100 nuclei were counted per experiment and nuclei with >5 foci were scored as positive. Data represent the mean ± s.e.m. of at least three independent experiments, and statistical analysis was performed using two-tailed paired t-tests.
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