Indirect immunofluorescence colocalization by microscopy was employed for the quantification of urinary podocytes and the expression of uPAR in the same cells. In this regard, cytologic urine smears obtained from the sediment were preincubated with rabbit nonimmune serum (PBS dilution 1: 100) in a humid chamber at room temperature for 1 h. Thereafter they were incubated with the two primary antibodies: anti-synaptopodin antibody and polyclonal anti-uPAR antibody (1: 200, Abcam) in a humid chamber at 4°C overnight. Three 5-min rinses with PBS were made, and the samples were incubated with the secondary antibodies: IgG anti-rabbit ALEXA Fluor 488® (1: 100, Abcam) for synaptopodin and IgG anti-mouse uPAR Alexa Fluor® 568 (1: 100) for uPAR in a humid chamber for 2 h at room temperature. Three 5-min rinses each were undertaken and were stained with DAPI. Samples were analyzed employing an epifluorescent microscope Nikon Eclipse E 200. The synaptopodin-uPAR-costained podocytes were counted in 10 randomly chosen 20× fields of the slides as de scribed above.
Anti rabbit igg alexa fluor 488
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-rabbit IgG Alexa Fluor 488 is a secondary antibody conjugated with Alexa Fluor 488 dye. It is designed to detect and visualize rabbit primary antibodies in various applications, such as immunofluorescence, flow cytometry, and Western blotting.
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Lab products found in correlation
Prolong gold antifade mountant, by Thermo Fisher Scientific (2 mentions)
Eclipse e200, by Nikon (1 mentions)
Anti synaptopodin, by Abcam (1 mentions)
Antifade mounting medium, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Ab22572, by Abcam (1 mentions)
Alexa fluor 647 anti goat igg, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Donkey serum, by Merck Group (1 mentions)
Goat serum, by Vector Laboratories (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
Anti mouse igg alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Saponin, by Merck Group (1 mentions)
α actinin, by Merck Group (1 mentions)
Lsm710, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (1 mentions)
Alexa fluor 594 anti mouse igg, by Abcam (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Citrate buffer ph 6, by Agilent Technologies (1 mentions)
Rabbit anti ki67, by Abcam (1 mentions)
17 protocols using anti rabbit igg alexa fluor 488
1
Quantifying Podocyte uPAR Expression
2
Quantifying Podocyturia: A Standardized Technique
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We have previously described the method to study podocyturia in detail [24 ]. Briefly, a mid-stream freshly voided urine sample was collected on-site after a minimum of 3 h without voiding; and 20 ml of urine were centrifuged at 700g for 5 min in a cytospin. The supernatant was discarded and the sediment was stored in 100 µl aliquots at room temperature mixed with a 1.5 ml solution of 40 % formaldehyde diluted in phosphate-buffered saline (PBS) (pH 7.2–7.4) to reach a final 10 % concentration. Podocyte nuclei were stained with 40,6-diamidino-2-phenylindole (DAPI). Podocytes were identified by immunofluorescence using anti-synaptopodin as the primary antibody (1:100, Abcam, Cambridge, MA, USA) and IgG anti-rabbit Alexa Fluor® 488 (1:100, Abcam, Cambridge, MA, USA) as the secondary antibody. Samples were analyzed employing an epifluorescent microscope. Following our standardized technique, podocytes were counted in 10 randomly chosen 20× fields and the average of the counted podocytes in the microscopy fields was considered as the final count for each subject. Results were corrected based on the urinary creatinine concentration of the initial urinary volume of 20 ml employed for podocyte counting [24 , 25 (link)].
3
Subcellular Localization of IGF2BP2
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The probe targeting HNF1A-S1 was designed by Ribo company (Guangzhou, China). Cells were fixed by 4% paraformaldehyde, and thencells were incubated with the probe at 37 °C overnight after permeabilization by PBS containing 0.5% Triton X-100 following the manufacturer’s instructions. The next day, cells were washed with SSC (Saline Sodium Citrate) buffer at 42 °C several times and incubated with anti-IGF2BP2 (Proteintech, 11601-1-AP, 1:200) overnight at 4 °C after blocking 30 minutes at room temperature. On the third day, the cells were washed and incubated with Alexa Fluor 488 anti-rabbit IgG (Abcam, 1:200, ab150077, Cambridge, UK). Nuclei were stained by DAPI, and then, the cells were pictured by confocal microscopy (LSM710, Jena, Thuringia, Germany).
4
6mA Immunofluorescence in Rat Cerebellum
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After temperature equilibration (10 min, RT), slides with cerebellum sections from two female and two male individual rats were rinsed with PBS 1x for 5 min, and underwent rehydration and permeabilization (PBS1X, Triton-X 0.3%), for 10 min. Slides were subjected to antigen retrieval (2M HCl) for 45 min and neutralization (0.1M Tris–HCl) for 20 min. After blocking for 1 h (PBS1x, BSA 1%, Triton-X 03%, RNAse A 50 μg/mL), slides were washed with 1X PBS (3 × 5 min) and incubated with primary antibody (1:500, rabbit-anti-6mA; Synaptic Systems, Gottingen, Germany) for 1 h (RT). After washing (3 × 5 min, 1x PBS) a 1:2000 Alexa Fluor 488-anti-rabbit IgG (ab150077; Abcam, Cambridge, United Kingdom) was used for visualization. Finally, the slides were washed with 1X PBS (3 × 5 min) and mounted with anti-fade mounting medium which contained DAPI (Thermo Fisher Scientific).
5
Immunostaining of Sox1 in Fixed Cells
6
Immunohistochemical Analysis of TIMP-1 Expression
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Murine HS and human keloid tissues were fixed and embedded in paraffin. For murine tissues, 4-μm-thick sections were first blocked with 10% donkey serum (Sigma-Aldrich, St. Louis, MO, USA) and then incubated with a polyclonal antibody against TIMP-1 (R&D Systems), washed, and incubated with secondary antibody (Alexa Fluor 647 anti-goat IgG; Thermo Fisher Scientific). For the human tissues, 4-μm-thick sections were first blocked with 10% goat serum (Vector Laboratories, Burlingame, CA, USA) and then incubated with a monoclonal antibody against TIMP-1 (Abcam), washed, and incubated with secondary antibody (Alexa Fluor 488 anti-rabbit IgG; Abcam). Finally, the sections were mounted in medium containing 4′,6-diamidino-2-phenylindole (Vectashield with DAPI; Vector Laboratories) and observed using an Olympus fluorescence microscope (Olympus).
7
Immunofluorescence Staining of Interphase Nuclei
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After cell fixation with 4% paraformaldehyde, the interphase nuclei were permeabilized with 0.2% Triton X100 for 8 min, were treated with 0.1% saponin (Sigma-Aldrich) for 12 min, and then washed twice in PBS for 15 min. A solution of 1% bovine serum albumin in PBS was used for blocking of non-specific binding of antibodies. The procedure was performed at room temperature (RT) for 1 h. After washing with PBS for 15 min, samples were incubated overnight at 4 °C with the monoclonal antibodies of interest: α-actinin (#A-7811, Sigma-Aldrich), H3K9ac (#06-942, Merc Millipore, MA, USA) and H4ac (#382160, Merc Millipore). The next day, the cells were washed twice in PBS for 5 min and incubated for 1 h with the appropriate secondary antibody conjugated with the fluorochrome of interest (#A11032, Alexa Fluor 594 anti-mouse IgG, Life Technologies Corporation, Eugene, OR, USA; #ab150077, Alexa Fluor 488 anti-rabbit IgG, Abcam, Cambridge, UK). Immuno-stained preparations were washed three times in PBS for 5 min, and DAPI (4′,6-diamidino-2-phenylindole dihydrochloride; #10236276001, Roche, Prague, CZ) was used for counterstaining the cell nuclei.
8
Double Immunofluorescence Labeling of SFTSV
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We performed double immunofluorescence labeling with cytokeratin-SFTSV and CD204-SFTSV on 4-μm thick tissue sections. We performed heat-mediated antigen retrieval for cytokeratin-SFTSV in pH 6.0 citrate buffer and for CD204-SFTSV antigen pH 9.0 Tris-EDTA buffer. After washing with PBS and blocking with 5% bovine serum albumin, we incubated the sections for 1 h at room temperature with a mixture of rabbit polyclonal anti-SFTSV antibody (diluted 1:1,000; TransGenic Inc., https://www.transgenic.co.jp ) and mouse monoclonal anti-CD204 (diluted 1:400; TransGenic Inc.) or mouse monoclonal anti-cytokeratin clone AE1/AE3 (diluted 1:200; Dako). After washing with PBS, we incubated the sections in a mixture of Alexa Fluor 488 anti-rabbit IgG (diluted 1:400; Abcam, https://www.abcam.com ), Alexa Fluor 594 anti-mouse IgG (diluted 1:400; Abcam), and DAPI (Dojindo, https://www.dojindo.com ). We then analyzed the tissue sections by using an LSM 710 (Leica, https://www.leicabiosystems.com ) confocal microscope.
9
Multimodal Tissue Imaging Techniques
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DiI detection. Frozen tissue sections (7 µm) were stained with Hoechst 33342 (Invitrogen, San Diego, CA, USA) for 20 min at room temperature.
hematoxylin and eosin staining. Paraffin-embedded sections (4 µm) were dewaxed, rehydrated, and stained with hematoxylin (Merck, Rahway, NJ, USA) for 10 min at room temperature, washed with tap water, and counterstained with eosin (Merck) for 5 min.
Immunofluorescence. Paraffin-embedded sections (4 µm) were dewaxed, rehydrated and antigen retrieval was carried out by heat-induced antigen retrieval treatment in 10 mM citrate buffer pH 6.0 (Dako, Fargo, ND, USA). Samples were incubated in 10% normal serum (rabbit or mouse) and 1% BSA in PBS for 1 h at room temperature, followed by incubation with the primary antibody in 1% BSA in PBS overnight at 4 °C (Rabbit Anti-Ki67, 5 µg/mL; Rabbit Anti-Collagen I, 5 µg/mL; Mouse Anti-Collagen III,1:600; Abcam, Cambridge, MA, United States). The sections were then washed and incubated with the secondary antibody (Anti-Rabbit IgG Alexa Fluor 488, 1:500; Anti-Mouse IgG Alexa Fluor 647, 1:500; Abcam) in 10% normal serum and 1% BSA in PBS for 1 h at room temperature in the dark. Samples were counterstained with Hoechst 33342, washed and embedded in a mounting medium (Permafluor, Thermo Scientific, Boston, MA, USA).
hematoxylin and eosin staining. Paraffin-embedded sections (4 µm) were dewaxed, rehydrated, and stained with hematoxylin (Merck, Rahway, NJ, USA) for 10 min at room temperature, washed with tap water, and counterstained with eosin (Merck) for 5 min.
Immunofluorescence. Paraffin-embedded sections (4 µm) were dewaxed, rehydrated and antigen retrieval was carried out by heat-induced antigen retrieval treatment in 10 mM citrate buffer pH 6.0 (Dako, Fargo, ND, USA). Samples were incubated in 10% normal serum (rabbit or mouse) and 1% BSA in PBS for 1 h at room temperature, followed by incubation with the primary antibody in 1% BSA in PBS overnight at 4 °C (Rabbit Anti-Ki67, 5 µg/mL; Rabbit Anti-Collagen I, 5 µg/mL; Mouse Anti-Collagen III,1:600; Abcam, Cambridge, MA, United States). The sections were then washed and incubated with the secondary antibody (Anti-Rabbit IgG Alexa Fluor 488, 1:500; Anti-Mouse IgG Alexa Fluor 647, 1:500; Abcam) in 10% normal serum and 1% BSA in PBS for 1 h at room temperature in the dark. Samples were counterstained with Hoechst 33342, washed and embedded in a mounting medium (Permafluor, Thermo Scientific, Boston, MA, USA).
10
Immunofluorescence Staining of Cell Cultures
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High (5 × 104 cells/cm2) and low (5 × 103 cells/cm2) density cell cultures were cultured for 4 days in basal media at 37 °C in 5 % CO2 in air before fixation in 70 % (v/v) ethanol for 15 min Fixed samples were permeabilised by incubation with 0.1 % Triton X-100 in PBS for 15 min before washing in PBS and blocking with pre-diluted goat serum (GeneTex, Irvine, CA, USA) for 30 min at 37 °C. Samples were then incubated with primary antibodies at dilutions between 1:50 and 1:250 overnight at 4 °C. Primary antibodies used were anti-cytokeratin 19 (FITC conjugated) (Santa Cruz Biotechnology, Dallas, TX, USA), anti-vimentin, anti-nestin and anti-fibronectin (all Abcam, Cambridge, UK). Non-conjugated primary antibodies were labelled with anti-rabbit IgG Alexa Fluor 488 (Abcam) for 30 min in the dark. Samples were then stained with DAPI for 15 min at room temperature to visualise nuclei before washing in PBS. Samples were then imaged using a Zeiss Axio Vert.A1 running Zen 2012 software (Carl Zeiss, Oberkochen, Germany).
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