Formalin-fixed and paraffin-embedded tissues were sectioned at 5 μm. Paraffin was removed by washing with xylene and then rehydrated with descending concentrations of ethanol. Antigen retrieval was performed by immersing the sections in an antigen retrieval buffer at 98.5 °C for 20 min. Slides were washed with PBS plus 0.1% triton (PBS-T) and blocked by block buffer [10% (vol/vol) donkey serum, 1% BSA in PBS] for 1 h at room temperature. The diluted antibody against Mac-3 (1:100, Cat#: 550292, BD, Franklin Lakes, NJ, USA) was placed as a drop on the slides and incubated overnight at 4 °C in a humidified chamber. The sections were then washed and covered with Alexa Fluor 555 Goat anti-Rat IgG (1:100, Invitrogen, Carlsbad, CA, USA) for 1 h in the dark. After washing with PBS three times, coverslips were mounted on slides using prolong gold anti-fade reagent (Invitrogen, Carlsbad, CA, USA). The fluorescent images were captured using a Nikon A1R Confocal Microscope (Tokyo, Japan).
Alexa fluor 555 goat anti rat igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 555 goat anti-rat IgG is a secondary antibody conjugate used for detection and visualization in immunoassays and other applications. It binds to rat primary antibodies and is labeled with the Alexa Fluor 555 fluorescent dye, which can be excited at 555 nm and emits light at 565 nm.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (5 mentions)
Alexa fluor 555 goat anti mouse igg, by Thermo Fisher Scientific (4 mentions)
Alexa fluor 555 goat anti rabbit igg, by Thermo Fisher Scientific (3 mentions)
Rat anti mouse f4 80 ab, by Bio-Rad (2 mentions)
Fitc conjugated goat anti mouse c3 ab, by MP Biomedicals (2 mentions)
Alexa fluor 488 goat anti rat igg, by Thermo Fisher Scientific (2 mentions)
Nb300 605, by Novus Biologicals (2 mentions)
Ab182422, by Abcam (2 mentions)
Ab133357, by Abcam (2 mentions)
Goat anti mouse igg hrp, by Thermo Fisher Scientific (2 mentions)
Gtx125868, by GeneTex (2 mentions)
Mca2314ga, by Bio-Rad (2 mentions)
Nbp2 61014, by Novus Biologicals (2 mentions)
Af690, by R&D Systems (2 mentions)
Goat anti mouse igg horseradish peroxidase hrp, by Thermo Fisher Scientific (2 mentions)
Ac033, by ABclonal (2 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
Goat anti rat igm alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
19 protocols using alexa fluor 555 goat anti rat igg
1
Immunofluorescence Analysis of Mac-3 Expression
2
Quantifying Kidney Complement C3 and Macrophages
3
Immunostaining of Cutaneous Squamous Cell Carcinoma
4
Quantifying Kidney Complement C3 and Macrophages
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Cryostat sections (4μm) of frozen kidneys were fixed with ice-cold acetone for 10min, and blocked with 10% goat serum to decrease background staining. FITC-conjugated goat anti-mouse C3 Ab (4.0 mg/ml, MP Biomedicals) was used directly at 1:500 dilution. Under ×200 magnification, 10 viewing fields from sections of each animal were photographed. Areas of positive staining were highlighted and the fluorescence intensity of C3 was determined using the plugin “Measure particles” of ImageJ software. F4/80-positive mononuclear phagocytes were visualized by staining with a rat anti-mouse F4/80 Ab (1.0 mg/ml, AbD seroTEC) used at 1:50 dilution followed by Alexa Fluor 555-goat anti-rat IgG (2.0 mg/ml, Invitrogen) used at 1:2000 dilution. Under 400x magnification, F4/80+ cells were counted by examining ten viewing fields randomly selected from the outer medulla on each slide.
5
Dopamine Transporter Immunofluorescence
6
Multimodal Immunohistochemical Profiling of Tissues
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Zebrafish embryos and rat tissues underwent cryosection at 5 to
7 µm and collected on gelatin-covered slides. Sections were then
processed for fluorescence immunohistochemistry. The primary antibodies were
anti-MMP9 (HPA001238, lot D104322, Sigma-Aldrich, USA, 1:100) for rat tissues,
anti-ZO1 (339100, lot 823765A, Invitrogen, USA, 1:100), anti-CD31 (DIA-310,
Dianova, Germany, 1:50), anti-laminin (Lama1, L9393, lot 103M4779,
Sigma-Aldrich, USA, 1:100), anti-collagen type IV (ab6586, lot GR193836-3,
Abcam, UK, 1:100), anti-Mmp9 (55345, lot OG2101, Anaspec, 1:50) for fish
embryos, and anti-phospho-p44/42 MAPK (Erk1/2) (4370, lot 15, Cell
Signaling Technology, 1:100) for fish embryos. The secondary antibodies were
Alexa Fluor 488 goat anti-rabbit IgG (A11008, lot 1705869, Invitrogen, USA,
1:200), Alexa Fluor 555 goat anti-mouse IgG (A21424, lot 1631208, Invitrogen,
USA, 1:200), Alexa Fluor 555 goat anti-rat IgG (A21434, lot 1670155, Invitrogen,
USA, 1:200), and Alexa Fluor 555 goat anti-rabbit IgG (A21428, lot 1608466,
Invitrogen, 1:200).
7 µm and collected on gelatin-covered slides. Sections were then
processed for fluorescence immunohistochemistry. The primary antibodies were
anti-MMP9 (HPA001238, lot D104322, Sigma-Aldrich, USA, 1:100) for rat tissues,
anti-ZO1 (339100, lot 823765A, Invitrogen, USA, 1:100), anti-CD31 (DIA-310,
Dianova, Germany, 1:50), anti-laminin (Lama1, L9393, lot 103M4779,
Sigma-Aldrich, USA, 1:100), anti-collagen type IV (ab6586, lot GR193836-3,
Abcam, UK, 1:100), anti-Mmp9 (55345, lot OG2101, Anaspec, 1:50) for fish
embryos, and anti-phospho-p44/42 MAPK (Erk1/2) (4370, lot 15, Cell
Signaling Technology, 1:100) for fish embryos. The secondary antibodies were
Alexa Fluor 488 goat anti-rabbit IgG (A11008, lot 1705869, Invitrogen, USA,
1:200), Alexa Fluor 555 goat anti-mouse IgG (A21424, lot 1631208, Invitrogen,
USA, 1:200), Alexa Fluor 555 goat anti-rat IgG (A21434, lot 1670155, Invitrogen,
USA, 1:200), and Alexa Fluor 555 goat anti-rabbit IgG (A21428, lot 1608466,
Invitrogen, 1:200).
7
Macrophage and Vessel Immunostaining in Wound Tissue
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Immunohistochemical staining was performed on 5 µm cryosections of day 7 wounds. Briefly, 5 µm cryosections were fixed either in 4% paraformaldehyde (CD144 and SMC vessels) or in acetone (F4/80 macrophages) and blocked with 5% goat serum. For macrophage staining primary antibodies, we used rat mAbs against F4/80(1:40, AbD serotec MCA497G, clone:CI: A3-1), rabbit mAbs against iNOS (1:800, Novus biologicals NBP1-33780, clone: K13-A), rat mAbs against TNFα (1: 10, AbD serotec MCA1488, clone: MP6-XT22), rabbit pAbs against CD206 (1:400, Santa Cruz sc-48758), and rabbit pAbs against Arginase (1:400, Novus biologicals NBP1-32731). For secondary antibodies, we used Alexa Fluor 488 goat anti-rat IgG(1:500, Invitrogen A11006), Alexa Fluor 555 goat anti-rat IgG(1:500, Invitrogen A21434), or Alexa Fluor 555 goat anti-rabbit IgG (1:500, Invitrogen A21428). For vessel staining, we used CD144 (1:40, BD pharmingen 550548) and FITC conjugated α-smooth muscle actin (α-SMA, 1:2000, Sigma-Aldrich F3777, clone:1A4). Immunofluorescent images were taken using a Zeiss microscope axiocam at ×200 magnification.
8
Angiogenesis Measurement in Wound Healing
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Immunohistochemical measurement of angiogenesis was performed at day 3 and day 7 postsurgery. After animal sacrifice, regenerated wounds were harvested, fixed in 4% neutral buffered formalin for 48 h, dehydrated with a gradient alcohol series, cleared in xylene, and embedded in paraffin. Tissue sections (5 μm) were deparaffinized, hydrated, and pretreated for antigen retrieval with citrate buffer. Sections were then incubated with a rat anti-mouse CD31 antibody (clone MEC 13.3, BD Pharmingen, 1 : 400) followed by exposure to Alexa Fluor 555 goat anti-rat IgG (Invitrogen, 1 : 500). Fluorescence was analyzed by conventional Zeiss Axioplan 2 Imaging microscopy.
Capillary density of healing area was determined by counting microvessels stained with isolectin B4 CD31 from at least 10 randomly selected fields/wound.
Capillary density of healing area was determined by counting microvessels stained with isolectin B4 CD31 from at least 10 randomly selected fields/wound.
9
Immunohistochemical Analysis of Mouse Eye Sections
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Mice were killed with an overdose of CO2, the eyes were immediately immersed in OCT medium, snap frozen in cooled isopentane on dry ice, and stored at −80°C until used. Primary antibody was placed for 1 hour at room temperature on 6-μm thick eye sections, followed by secondary biotinylated rabbit anti-rat (DakoCytomation, Glostrup, Denmark) or biotinylated mouse anti-hamster Ig cocktail (BD Pharmingen, Oxford, UK) for 30 minutes at room temperature followed by Vectastain ABC-AP kit (Vector Laboratories, Peterborough, UK) for 30 minutes. For confocal imaging secondary fluorescent-labelled antibodies Alexa Fluor 555 goat anti-rat IgG (Invitrogen, Waltham, MA, USA) and Alexa Fluor 488 goat anti-rat IgG (Invitrogen) were used together with nuclear staining 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Peterborough, UK). The following primary monoclonal antibodies (mAb) were used in concentrations as suggested by the manufacturer: rat anti-mouse CD11b (clone M1/70); rat anti-mouse CD4 (clone H129.19), rat anti-mouse CD8α (clone 53-6.7), rat anti-mouse Gr-1 (clone RB6-8C5), hamster anti-mouse CD11c (clone HL3; all from BD Pharmingen) and rat anti–mouse F4/80 (clone C1:A3-1; AbD Serotec, Kidlington, UK).
10
Immunostaining of Brain Sections
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Brain sections were washed in PBS three times, permeabilized, and blocked with PBS containing 5%(wt/vol) BSA and 0.3% Triton X-100 for 30 min at room temperature, and incubated with primary antibody at 4 °C overnight. Sections were subsequently washed in PBS, incubated with a secondary antibody for 1h at room temperature, and washed with PBS. The following antibodies and reagents were used for immunostaining: anti-BLBP (Abcam, AB32423, 1:1000), anti-PAX6 (Covance, 901301, 1:200), anti-Tbr2 (Abcam, AB183991, 1:200), anti-CTIP2 (Abcam, AB18465, 1:500), anti-GFAP (Millipore, NE1015, 1:500), anti-prox1 (Millipore, MAB5654, 1:100), anti-NeuN (Millipore, MAB377B, 1:100), anti-Ki67 (Abcam, AB15580, 1:500), anti-caspase3 (Cell Signaling Technology, P42574, 1:500), Alexa Fluor 555 goat anti-rat IgG (Invitrogen, A31570, 1:1000); Alexa Fluor 555 goat anti-mouse IgG (Invitrogen, A21050, 1:1000); Alexa Fluor 488 goat anti-mouse IgG (Invitrogen, A11008, 1:1000), Alexa Fluor 555 donkey anti-rabbit IgG (Invitrogen, A31572, 1:1000); and Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, A11006, 1:1000), EdU imaging Kit (Thermo Fisher, C10340), Hoechst (Invitrogen 33342). Sections were imaged with a confocal microscope (Olympus FV1200, Japan), and analyzed by ImageJ software.
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