The following primary antibodies were used for immunohistochemistry experiments with indicated final concentrations: mouse monoclonal anti-ppERK1/2 (M9692, 1:1000/1.5-2 μg/mL, Sigma), rabbit monoclonal anti-β-Catenin (#9562,1:200, CST), chicken monoclonal IgY anti-GFP (A10262, 5 μg/mL, Invitrogen), rat anti-HA (#3K10, 1:400, Roche), mouse anti-F59 Myosin Heavy Chain (AB528373, 1:10/ 0.2-0.5 μg/mL, DSHB), and rabbit anti-pFAK (44-624G, 1:400, Invitrogen). Primary antibodies were targeted with 1:200 dilutions of Alexa Fluor 488 goat anti-chicken IgG H+L (A11039, Invitrogen), Alexa Fluor 594 goat anti-mouse IgG H+L (A11005, Invitrogen), Alexa Fluor 647 goat anti-rabbit IgG (A21245, Invitrogen) or Alexa Fluor 647 goat anti-rat IgG H+L (A21247, Invitrogen) for corresponding primary species. Alexa Fluor 488 Phalloidin (A12379, 1:200, Thermo Fisher) was used against F-actin of muscle fibers.
Alexa fluor 647 goat anti rat igg h l
Manufactured by Thermo Fisher Scientific
Alexa Fluor 647 goat anti-rat IgG H+L is a secondary antibody conjugated with the Alexa Fluor 647 fluorescent dye. It is designed to detect and bind to the heavy and light chains of rat immunoglobulin G (IgG) antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 594 goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
M9692, by Merck Group (1 mentions)
Alexa fluor 488 goat anti chicken igg h l, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Mouse seroblock fcr, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Ab183032, by Abcam (1 mentions)
Ab31940, by Abcam (1 mentions)
Ab97959, by Abcam (1 mentions)
Ab18465, by Abcam (1 mentions)
Ab6326, by Abcam (1 mentions)
Kihc 5, by Proteintech (1 mentions)
Alexa fluor 488 goat anti mouse igg1, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 donkey anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 goat anti mouse igg1, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Csu w1 confocal unit, by Olympus (1 mentions)
μ slide 8 well, by Ibidi (1 mentions)
5 protocols using alexa fluor 647 goat anti rat igg h l
1
Immunohistochemistry Antibody Staining Protocol
2
Membrane CD14 and P2X7 Expression
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For membrane CD14 flow cytometry, BMDMs seeded in 24-well plates were washed and incubated for 30 min at 37°C in E-total buffer supplemented with or without 5 mM of ATP, in presence or absence of P2X7 receptor antagonist A438079 (10 μM). To stain surface CD14, cells were washed and incubated with mouse seroblock FcR (BD biosciences) and then stained with anti-mouse CD14 (clone rmC5-3; 553738; BD biosciences; RRID:AB_395020 ) for 30 min at 4°C. Cells were washed again and incubated with secondary Alexa Fluor 647 goat anti-rat IgG (H+L) (A21247; Invitrogen, RRID:AB_141778 ) for an additional 30 min at 4°C. Finally, cells were washed and fixed with 4% PFA in PBS and then scrapped and aliquoted in flow cytometry tubes. For human P2X7 flow cytometry, monocytes were determined from peripheral blood mononuclear cells from non-septic and septic patients by CD3− CD14+ selection, and P2X7 receptor surface expression was determined using the monoclonal anti-P2X7 L4 clone (Buell et al., 1998 (link); Martínez-García et al., 2019 (link)). All samples were subjected to flow cytometry analysis using a BD FACSCanto flow cytometer (BD) and FACSDiva software (BD, RRID:SCR_001456 ) by gating for BMDM cells based on FSC versus SSC parameters.
3
Immunolabeling of Mouse Brain Sections
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PBS-perfused brains were fixed in 4% paraformaldehyde, and coronal sections were prepared for IFA (thickness: E18.5, 20 μm; postnatal, 30 μm) or IHC and histochemistry (thickness: 3 μm). Mouse monoclonal antibodies against MCMV IE1 (51 (link)); rabbit monoclonal antibodies against SOX2 (Abcam, ab97959), Tbr1 (Abcam, ab31940, ab183032), Ctip2 (Abcam, ab240636), GFAP (Proteintech, 16825-1-AP), Iba1 (Abcam, ab178847), GSDMD (Affinity, AF4012), or CC3 (Cell Signaling Technology, 9661); and rat monoclonal antibodies against BrdU (Abcam, ab6326) or Ctip2 (Abcam, ab18465) were used in IFA or IHC as indicated. Secondary antibodies for IFA included Alexa Fluor 488 goat anti–mouse IgG1 (Invitrogen, A-21121), Alexa Fluor 568 donkey anti–rabbit IgG (H+L) (Invitrogen, A-10042), Alexa Fluor 647 goat anti–mouse IgG1 (Invitrogen, A-21240), and Alexa Fluor 647 goat anti–rat IgG (H+L) (Invitrogen, A-21247). DAPI (Life Technologies) was used for nuclei counterstaining. Horseradish peroxidase–conjugated antibody (Proteintech, KIHC-5) was used for IHC. IFA, IHC, and histochemistry were performed as described previously (52 (link)).
4
Quantitative RNA and Protein Analysis
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Trypsinized cells were transferred onto μ-Slide 8-well (Ibidi, Martinsried, Germany) coated with Laminin-511 and cultured for 1 h at 37°C and 5% CO2. Then, cells were fixed and subjected to smFISH as described above using Nanog exonic probes. After the image acquisition, cells were subjected to immunostaining, as described in the immunostaining section above, using the anti-Nanog antibody and Alexa Fluor 647 goat anti-rat IgG (H+L) (1:500, Life Technologies). The images were acquired using an Olympus IX83 microscope with a CSU-W1 confocal unit, 60× Olympus oil-immersion objective of 1.42 NA; 101 z planes per site, spanning 15 μm, were analyzed. smFISH images were filtered using ImageJ, with a one-pixel diameter 3D median filter, and subjected to background subtraction via a rolling ball radius of 2 pixels. Further fluorescence subtraction (100, 16-bit pixel unit) for almost complete removal of background intensity and maximum-intensity projection was subsequently performed. Immunostained images were filtered with a two-pixel diameter 3D median filter, and subjected to fluorescence subtraction (300, 16-bit pixel unit) for almost complete removal of background intensity and maximum-intensity projection, using ImageJ. The freehand selection tool of ImageJ was used for measurements of integrated signal intensity in each cell.
5
Embryonic Cell Lineage Tracing
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Embryos still in decidua were fixed in 4% Paraformaldehyde (PFA) for two hours, before they were soaked in 30% sucrose and mounted in OCT compound. 7 µm sections were prepared using a cryostat. The sections were streptavidin/biotin blocked followed by serum blocking (PBS with 10% FCS, 0.05% Tween20 and of 10% goat serum (DAKO) for 1 hour before the sections were incubated with primary antibodies at 4 °C overnight in blocking buffer. Primary antibodies used were rabbit anti –GFP (598, polyclonal, MBL) (1/200); rat anti-mouse c-kit (553352, clone 2B8, BD Biosciences) (1/100); biotinylated rat anti-mouse Tie2 (13–5987, clone TEK4 eBioscience) (1/100). After staining, the sections were washed three times in PBST for 15 minutes each and then incubated with fluorochrome-conjugated secondary antibody at room temperature for 1 hour. These were Alexa Fluor® 488 Donkey Anti-Rabbit IgG (H + L) (A21206, Life Technologies); Alexa Fluor® 647 Goat Anti-Rat IgG (H + L) (A21247, Life Technologies); Streptavidin, Alexa Fluor® 555 Conjugate (S32355, Life Technologies). All secondary antibodies were used at 1/400 dilution. The sections were then further washed three times in PBS and mounted using Prolong Gold anti-fade medium with DAPI (Life Technologies). Images were taken using a low-light time lapse microscope (Leica) using the Metamorph imaging software and processed using ImageJ.
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