Anal tubes were isolated and fixed in ice-cold acetone for 10 min, then washed with PBS overnight at 4 °C and rewashed for 4 h with a change of PBS per hour. Cryosections with a 10-μm thickness were used. The non-specific binding of primary antibodies was blocked by incubation with PBS containing 1% BSA and 5% non-immune goat serum for 1 h. Incubation was carried out overnight at 4 °C with a rabbit polyclonal antibody to TMEM16A (ab53212, abcam) diluted 1:200, together with a rat anti-c-Kit antibody (ACK2, Chemicon, 1:100) or mouse monoclonal Myh11 antibody (ab683 clone 1G12, 1:200; abcam). After washing in PBST, sections were incubated with a Alexa Fluor 555-conjugated goat anti-Rabbit antibody (Sigma) diluted 1:250 and a FITC-conjugated goat anti-rat antibody (Invitrogen) diluted 1:250 or a Alexa Fluor 488–conjugated goat anti-mouse antibody (Cell signaling technology) for 1 h. 4,6-diamidino-2-phenylindole was used for nuclear staining. Immunoreactivity was evaluated using a FV1000 confocal laser scanning microscope system (Olympus).
Alexa fluor 488 conjugated goat anti mouse antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Alexa Fluor 488-conjugated goat anti-mouse antibody is a secondary antibody that is conjugated to the fluorescent dye Alexa Fluor 488. It is designed to detect and bind to primary antibodies raised in mice.
Automatically generated - may contain errors
Lab products found in correlation
Ab53212, by Abcam (1 mentions)
Fv1000 confocal laser scanning microscope system, by Olympus (1 mentions)
Ab683 clone 1g12, by Abcam (1 mentions)
Cleaved caspase 3, by Merck Group (1 mentions)
α tubulin, by Merck Group (1 mentions)
Epiquik in situ dna damage assay kit, by Epigentek (1 mentions)
Horseradish peroxidase hrp conjugated goat anti rabbit antibody, by Cell Signaling Technology (1 mentions)
Nf κb, by Merck Group (1 mentions)
Dna damage quantification colorimetric kit, by Abcam (1 mentions)
Hrp conjugated goat anti mouse antibody, by Cell Signaling Technology (1 mentions)
Cleaved parp, by Merck Group (1 mentions)
Cleaved caspase 7, by Cell Signaling Technology (1 mentions)
Cleaved caspase 9, by Cell Signaling Technology (1 mentions)
Dcfh da, by Merck Group (1 mentions)
Cisplatin, by Merck Group (1 mentions)
Traf6, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Proteintech (1 mentions)
A1933, by Merck Group (1 mentions)
Influx flow cytometer, by BD (1 mentions)
B900780, by Proteintech (1 mentions)
4 protocols using alexa fluor 488 conjugated goat anti mouse antibody
1
Immunolocalization of TMEM16A in Anal Tissue
2
Apoptosis and DNA Damage Assessment
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The FITC-labelled Annexin V Apoptosis Detection Kit was obtained from BD Biosciences (San Jose, CA, USA). DNA Damage Quantification Colorimetric Kit was purchased from Biovision (Milpitas, CA, USA) and EpiQuik In Situ DNA Damage Assay kit was obtained from Epigentek (Farmingdale, NY, USA). JC-1, cisplatin, and DCFH-DA were purchased from Sigma-Aldrich (St. Louis, MO, USA). The culture medium was obtained from Invitrogen Technologies (Carlsbad, CA, USA). The antibodies were obtained from the following sources: TRAF6 and MVP from Abcam (Cambridge, UK); α-tubulin from Sigma-Aldrich; p62, NFκB, cleaved-PARP, cleaved-caspase 3, cleaved-caspase 7, cleaved-caspase 9, IKKβ, IKKα, phosphor-IKKβ, horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody, HRP-conjugated goat anti-mouse antibody, and Alexa Fluor 488-conjugated goat anti-mouse antibody from Cell Signalling Technology (Danvers, MA, USA).
3
Immune Cell Profiling in CSF and PBL
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All operations in this section were performed at low temperature. To evaluate PMN and MN populations, processed CSF cells or PBLs were stained with 1 μg/ml Hoechst 33342 for 5 min, then fixed in PBS containing 1% paraformaldehyde, and checked through an Influx flow cytometer (BD). For cell immunostaining, processed CSF cells or PBLs were blocked for 15 min in PBS containing 0.5% bovine serum albumin (Sigma-Aldrich, A1933) and 3% FBS and then incubated for 30 min with anti-VSIG4 (4 μg/ml, Santa Cruz, sc-53977), anti-CD1C (2 μg/ml, NOVUS, NBP2-62220), or equal concentrations of isotype-matched control antibodies. After washing three times with PBS containing 3% FBS, the suspension was incubated with 2 μg/ml Alexafluor 488-conjugated goat anti-mouse antibody (4408S, Cell Signaling) for 30 min in PBS containing 0.5% bovine serum albumin and 10% goat serum (B900780, Proteintech) and then incubated for another 5 min with 1 μg/ml Hoechst 33342. The cells were washed three times and fixed in PBS containing 1% paraformaldehyde. Cellular fluorescence was measured using an Influx flow cytometer, and data were analyzed using FlowJo software.
4
Immunofluorescence Staining of RAA Tissues
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The frozen RAA tissues were embedded in the optimum cutting temperature (OCT) compound and sectioned at −25°C in a cryostat. Sections (5 μm in thickness) were fixed with 4% polyformaldehyde for 15 minutes at room temperature. The sections were permeabilized with 0.1% TritonX-100 for 20 minutes followed by blocking with 10% goat serum for 1 hour at room temperature. Then, the sections were incubated with anti-Cox IV antibody (Cell Signaling Technology, Danvers, MA, USA) diluted at 1 : 200, anti-LAMP-1 antibody (Cell Signaling Technology, Danvers, MA, USA) diluted at 1 : 200, or anti-LC3B antibody (Abcam) diluted at 1 : 100 overnight at 4°C. Subsequently, the sections were incubated with Alexa Fluor 488-conjugated goat anti-mouse antibody (Cell Signaling Technology, Danvers, MA, USA) diluted at 1 : 500, or Alexa Fluor 594-conjugated goat anti-rabbit antibody (Cell Signaling Technology, Danvers, MA, USA) diluted at 1 : 500 for 1 hour at room temperature. DAPI (Solarbio Science & Technology, Beijing, China) was used to stain the cell nuclei. Finally, the fluorescence images were captured by using a laser scanning microscope and further analyzed by ImagePro Plus 6.0 (Media Cybernetics, Inc., Bethesda, MD, USA).
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