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Species specific fluorescent secondary antibodies

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Species-specific fluorescent secondary antibodies are laboratory reagents used to detect and visualize target proteins in biological samples. These antibodies are designed to bind to the constant region of primary antibodies raised in a specific animal species, and are conjugated with fluorescent dyes that emit light when excited. This allows for the indirect detection and localization of the target proteins through fluorescence microscopy or other imaging techniques.

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Lab products found in correlation

Tsl aobs sp5, by Leica (3 mentions) Vibratome, by Leica (3 mentions) Ti e inverted microscope, by Nikon (3 mentions) Anti tubb3 antibody β tubulin 3 antibody, by Fortrea (2 mentions) Tsl aobs sp5 confocal microscope, by Leica (1 mentions) Anti gfap antibody, by Thermo Fisher Scientific (1 mentions) Evos fl auto fluorescent microscope, by Thermo Fisher Scientific (1 mentions) Sc 372, by Santa Cruz Biotechnology (1 mentions) Tubb3, by Thermo Fisher Scientific (1 mentions) Tubb3, by Fortrea (1 mentions) Stellaris confocal microscope, by Leica (1 mentions) Ab196436, by Abcam (1 mentions) Sc 74470, by Santa Cruz Biotechnology (1 mentions) Gtx118619, by GeneTex (1 mentions) Hoechst 33342, by Thermo Fisher Scientific (1 mentions) Rabbit anti neurofilament 200, by Merck Group (1 mentions) Rat anti f4 80, by Abcam (1 mentions) Goat anti myelin basic protein, by Santa Cruz Biotechnology (1 mentions) Chicken anti p0, by Aves Labs (1 mentions) Chicken anti gfap, by Aves Labs (1 mentions)

Close spelling variants of this product

Species‐specific secondary antibodies (8 citations) Specific antibodies (8 citations) Specific fluorescent secondary antibodies (2 citations) Suitable species-specific fluorescent secondary antibodies (2 citations)

13 protocols using species specific fluorescent secondary antibodies

1

RGC Immunocytochemistry Protocol

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Cultured primary RGCs were fixed in 4% PF and blocked in buffer containing 5% donkey serum, 0.15% Tween-20 and 1xPBS (pH 7.4). Cells were then incubated with anti-Tubb3 antibody (β-tubulin III antibody, 1:500; Covance, Princeton, NJ), anti-Ripk1 and anti-Ripk3 antibodies (1:250; both from GeneTex, Inc, Irvine, CA), followed by species-specific secondary fluorescent antibodies (Life Technologies, Grand Island, NY). Negative controls were incubated with secondary antibody only. Imaging was performed with a confocal microscope (Leica TSL AOBS SP5; Leica Microsystems, Exton, PA).
Dvoriantchikova G., Degterev A, & Ivanov D. (2014). Retinal ganglion cell (RGC) programmed necrosis contributes to ischemia-reperfusion-induced retinal damage. Experimental eye research, 123, 1-7.
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2

Retinal Immunohistochemistry Protocol

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Fixed retinas were sectioned to a thickness of 100 μm with a vibratome (Leica Microsystems, Exton, PA) and immunostained as described earlier (Dvoriantchikova et al., 2009a ; Dvoriantchikova et al., 2009b (link); Dvoriantchikova et al., 2010b (link)). Briefly, sections were permeabilized with 0.3% Triton X-100 in PBS for one hour, rinsed three times in PBS, blocked in buffer (5% donkey serum, 2% BSA and 0.15% Tween-20 in PBS) for 1 hour and incubated overnight with anti-Ripk1 (1:250) and anti-Ripk3 (1:250) specific antibodies (both from GeneTex, Inc, Irvine, CA) as well as anti-Tubb3 antibody (β-tubulin III antibody, 1:500; Covance, Princeton, NJ), followed by species-specific secondary fluorescent antibodies (Life Technologies, Grand Island, NY). Control sections were incubated without primary antibodies. Imaging was performed with a Leica TSL AOBS SP5 confocal microscope (Leica Microsystems, Exton, PA).
Dvoriantchikova G., Degterev A, & Ivanov D. (2014). Retinal ganglion cell (RGC) programmed necrosis contributes to ischemia-reperfusion-induced retinal damage. Experimental eye research, 123, 1-7.
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3

Notch1 Expression Analysis in Cells

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Notch1+ cells were placed on cover slips, fixed after 30 minutes in 4% PF, and blocked with 5% normal donkey serum with 0.15% Tween-20 in PBS at pH 7.4. Cells were then incubated with Notch1 primary antibody (130-101-868, Miltenyi Biotec Inc., San Diego, CA) followed by species-specific secondary fluorescent antibodies (Invitrogen, Carlsbad, CA). Negative controls were incubated with secondary antibodies only. Imaging was performed with a confocal microscope (Leica TSL AOBS SP5; Leica Microsystems). Propidium iodide (PI) was used to visualize the nucleus of the cell. Individual cover slips were sampled randomly to collect a total of 10 images using a 20X objective lens. The Notch1 positive cells and PI positive cells (the total number of cells) were counted using ImageJ software. The percentage of Notch1 positive cells relative to the total number of cells was determined.
Dvoriantchikova G., Perea-Martinez I., Pappas S., Barry A.F., Danek D., Dvoriantchikova X., Pelaez D, & Ivanov D. (2015). Molecular Characterization of Notch1 Positive Progenitor Cells in the Developing Retina. PLoS ONE, 10(6), e0131054.
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4

Immunofluorescence Imaging of RPE Cells

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RPE sheets were fixed in 4% PFA and blocked with 5% normal donkey serum with 0.15% Tween-20 in PBS at pH 7.4. Cells were then incubated with the primary antibody (Supplementary Data S10) followed by species-specific secondary fluorescent antibodies (Invitrogen, Carlsbad, CA). Negative controls were incubated with the secondary antibody only. Imaging was performed with a confocal laser microscope (Leica TSL AOBS SP5; Leica Microsystems).
Dvoriantchikova G., Seemungal R.J, & Ivanov D. (2019). The epigenetic basis for the impaired ability of adult murine retinal pigment epithelium cells to regenerate retinal tissue. Scientific Reports, 9, 3860.
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5

Immunohistochemistry for Retinal Glial Markers

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Immunohistochemistry for Glast (Slc1a3) and Gfap was performed as described previously [20 (link)]. Briefly, the fixed retinas were sectioned to a thickness of 100 μm with a Vibratome (Leica Microsystems) and immunostained with the anti-Glast (ACSA-1) antibody (130–118-984, Miltenyi Biotec, Auburn, CA), the anti-Gfap antibody (Table 1), and species-specific secondary fluorescent antibodies (Thermo Fisher Scientific, Carlsbad, CA). Control sections were incubated without primary antibodies. Imaging was performed with a Leica TSL AOBS SP5 confocal laser microscope (Leica Microsystems).
Dvoriantchikova G., Seemungal R.J, & Ivanov D. (2019). Development and epigenetic plasticity of murine Müller glia. Biochimica et biophysica acta. Molecular cell research, 1866(10), 1584-1594.
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6

Fluorescent Immunostaining of RGCs and AAV-293 Cells

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Primary RGCs and AAV-293 cells were fixed in 4% paraformaldehyde (PFA) for 30 minutes, rinsed in PBS (3 × 10 minutes) and blocked with 5% normal donkey serum with 0.15% Tween-20 in PBS at pH 7.4. Cells were then incubated with either both beta III Tubulin antibody (Tubb3, 1:250, Covance, Denver, PA) and p65 antibody (1:400; sc-372, Santa Cruz Biotechnology, Dallas, TX) or with Tubb3 alone, followed by species-specific secondary fluorescent antibodies (Thermo Fisher Scientific, Grand Island, NY). Negative controls were incubated with secondary antibodies only. The cells were imaged using an EVOS FL Auto fluorescent microscope with onstage incubation capabilities (Thermo Fisher Scientific, Grand Island, NY).
Dvoriantchikova G., Pappas S., Luo X., Ribeiro M., Danek D., Pelaez D., Park K.K, & Ivanov D. (2016). Virally delivered, constitutively active NFκB improves survival of injured retinal ganglion cells. The European journal of neuroscience, 44(11), 2935-2943.
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7

Immunofluorescent Analysis of Retinal Proteins

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Eyes were enucleated, fixed with 4% paraformaldehyde in PBS, washed with PBS, and then retinas were removed. The fixed retinas were sectioned with a vibratome (Leica Microsystems,US) to a thickness of 100 μm, and, then, sections were permeabilized with 0.3% Triton X-100/PBS for 1 h, washed three times with PBS, blocked in a buffer (5% donkey serum, 2% bovine serum albumin and 0.15% Tween-20 in PBS) for 1 h, and incubated overnight with Mlkl (phospho S345) antibody (p-Mlkl, 1:300, ab196436, Abcam, US), Steap3 antibody (1:100, pHyde, sc-376327, Santa Cruz Biotechnology, US), Parp1 antibody (1:100, sc-74470, Santa Cruz Biotechnology, US), Rbpms antibody (1:400, GTX118619, GeneTex, US), and Tubb3 antibody. The next day, the retinas were washed three times with PBS, and incubated with species-specific secondary fluorescent antibodies (ThermoFisher Scientific, US). Control sections were incubated without primary antibodies. Imaging was performed with Leica STELLARIS confocal microscope (Leica Microsystems, US).
Dvoriantchikova G., Lypka K.R., Adis E.V, & Ivanov D. (2022). Multiple types of programmed necrosis such as necroptosis, pyroptosis, oxytosis/ferroptosis, and parthanatos contribute simultaneously to retinal damage after ischemia–reperfusion. Scientific Reports, 12, 17152.
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8

Characterizing Spinal Cord Tissue Composition

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Isolated spinal cords were flash frozen, and then cryosectioned transversely (8 week tissue) or longitudinally (2 week tissue) in 18 μm sections. Samples were fixed, permeabilized as necessary, and incubated overnight at 4°C with primary antibodies. The following antibodies were used for primary detection: rat anti-F4/80 (1:200, Abcam, Cambridge, United Kingdom), goat anti-arginase (1:100, Santa Cruz, Dallas, TX, USA), rabbit anti-neurofilament-200 (1:200, Sigma), goat anti-myelin basic protein (MBP; 1:500, Santa Cruz), chicken anti-P0 (1:250, Aves Labs, Tigard, OR), chicken anti-GFAP (1:1000, Aves Labs). Species-specific fluorescent secondary antibodies were used for detection at 1:1000 (Life Technologies, Carlsbad, CA, USA). Hoechst 33342 (Life Technologies) was used as a counterstain in all tissue sections. Immunostained tissue sections were imaged using an AxioObserver inverted fluorescent microscope (Zeiss) using a 10× dry objective.
Dumont C.M., Carlson M.A., Munsell M.K., Ciciriello A.J., Strnadova K., Park J., Cummings B.J., Anderson A.J, & Shea L.D. (2019). Aligned hydrogel tubes guide regeneration following spinal cord injury. Acta biomaterialia, 86, 312-322.
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9

Immunofluorescence Staining Protocol with Pre-Extraction

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Cells were washed in PBS and fixed in 4% paraformaldehyde for 20 min; cells were then extracted in PBS-0.5% Triton X-100 for 5 min, washed 3 times with PBS, blocked for 30 min in PBS containing 3% BSA (PBS-BSA) and incubated with primary antibodies diluted in PBS-BSA for 60 min. Samples were washed 3 times for 5 min with PBS-0.05% Triton X-100 and primary antibodies were detected using species-specific fluorescent secondary antibodies (Life Technologies). Samples were washed 3 more times for 5 min with PBS-0.05% Triton X-100 prior to DNA detection (2.5 μg/ml Hoechst). For pre-extraction, cells were washed once with PBS, and then incubated in CSK buffer (100 nM NaCl, 300 mM sucrose, 3 mM MgCl2 and 10 mM PIPES pH 6.8) containing 0.5% Triton X-100 for 5 minutes on ice. Cells were then washed 3 times with PBS, fixed and processed as above.
Zhang C.Z., Spektor A., Cornils H., Francis J.M., Jackson E.K., Liu S., Meyerson M, & Pellman D. (2015). CHROMOTHRIPSIS FROM DNA DAMAGE IN MICRONUCLEI. Nature, 522(7555), 179-184.
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10

Immunofluorescence Staining Protocol with Pre-Extraction

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Cells were washed in PBS and fixed in 4% paraformaldehyde for 20 min; cells were then extracted in PBS-0.5% Triton X-100 for 5 min, washed 3 times with PBS, blocked for 30 min in PBS containing 3% BSA (PBS-BSA) and incubated with primary antibodies diluted in PBS-BSA for 60 min. Samples were washed 3 times for 5 min with PBS-0.05% Triton X-100 and primary antibodies were detected using species-specific fluorescent secondary antibodies (Life Technologies). Samples were washed 3 more times for 5 min with PBS-0.05% Triton X-100 prior to DNA detection (2.5 μg/ml Hoechst). For pre-extraction, cells were washed once with PBS, and then incubated in CSK buffer (100 nM NaCl, 300 mM sucrose, 3 mM MgCl2 and 10 mM PIPES pH 6.8) containing 0.5% Triton X-100 for 5 minutes on ice. Cells were then washed 3 times with PBS, fixed and processed as above.
Zhang C.Z., Spektor A., Cornils H., Francis J.M., Jackson E.K., Liu S., Meyerson M, & Pellman D. (2015). CHROMOTHRIPSIS FROM DNA DAMAGE IN MICRONUCLEI. Nature, 522(7555), 179-184.
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