Dissected mammary fat pads were fixed in MethaCarn and embedded in paraffin. Seven μm-thick sections were deparaffinized before staining with primary antibodies (overnight, 4°C), and secondary antibodies (1 h, room temperature). Nuclei were counterstained with DAPI. Primary antibodies used were: rabbit polyclonal anti-PAR3 (1:200; Chemicon), anti-aPKC (1:200; clone C-20, Santa Cruz Biotechnology), anti-RAB11A (1:200; Abcam), anti-pSTAT5 (Tyr694, 1:100; Cell Signalling), anti-cleaved caspase 3 (1:100; Cell Signalling), anti-WAP (1:300; clone R-131, Santa Cruz Biotechnology) and anti-keratin 5 (K5) (1:2,000; Covance); rabbit monoclonal anti-KI67 (1:100; clone SP6, Neo Markers); and mouse monoclonal anti-HTT (1:300; 4C8), anti-E-cadherin (1:200; BD Bioscience) and anti-GM130 (1:100; BD Bioscience). Antigen retrieval was performed by boiling the slides for 10 min in a microwave in 10 mM citrate buffer (pH 6) for cleaved caspase 3, Ki67, WAP, and p-STAT5A, or in EDTA buffer (pH 8.8) for 10 min for PAR3, aPKC, RAB11A, HTT, GM130, K5, and E-cadherin antibodies. Secondary antibodies used were goat anti-mouse and anti-rabbit conjugated to AlexaFluor-488 or AlexaFluor-555 or Biotin (Vector Laboratories).
Alexa fluor 488
Manufactured by Vector Laboratories
Sourced in United States
Alexa Fluor 488 is a fluorescent dye produced by Vector Laboratories. It is a bright, photostable dye with excitation and emission maxima at 495 nm and 519 nm, respectively. Alexa Fluor 488 can be used for a variety of labeling applications, including immunohistochemistry, flow cytometry, and fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 546, by Vector Laboratories (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Clone c 20, by Santa Cruz Biotechnology (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti apkc, by Santa Cruz Biotechnology (1 mentions)
Anti rab11a, by Abcam (1 mentions)
Anti pstat5 tyr694, by Cell Signaling Technology (1 mentions)
Anti wap, by Santa Cruz Biotechnology (1 mentions)
Anti e cadherin, by BD (1 mentions)
Anti gm130, by BD (1 mentions)
Lsm 510 confocal microscope, by Zeiss (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Alexa fluor 594, by Vector Laboratories (1 mentions)
Neurobiotin, by Vector Laboratories (1 mentions)
Dylight549 conjugated streptavidin, by Jackson ImmunoResearch (1 mentions)
Goat anti mouse igg conjugated to alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Olig2, by Merck Group (1 mentions)
Goat anti rabbit igg conjugated to alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Lycopersicon esculentum tomato lectin, by Vector Laboratories (1 mentions)
Glial fibrillary acidic protein (gfap), by Merck Group (1 mentions)
7 protocols using alexa fluor 488
1
Immunofluorescence analysis of mammary gland
2
Immunolocalization of PRG4 in TMJ Discs
3
Immunofluorescent Labeling of Prdx6 in Brain Tissue
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Cryopreserved frozen brain tissues were prepared by cutting 10 μm thick coronal sections that were 1.5 mm rostral to the bregma using a cryostat. Prdx6 labeling was performed by incubating sections with anti-Prdx6 rabbit polyclonal antibody (Abcam American; 1 : 100 dilution) and mouse anti-Neuronal Nuclei monoclonal antibody (NeuN, 1 : 500; Millipore) overnight at 4°C. After washing in phosphate-buffered saline (PBS), sections were incubated with green-fluorescent Alexa Fluor 488 and red-fluorescent Alexa Fluor 594 (1 : 500 for both; Vector Laboratories, Inc., Burlingame, CA) for 1 hour at room temperature. All sections were observed and the images were captured using a fluorescence microscope (BX51, Olympus, Tokyo, Japan).
4
Dye Injection and Labeling of AII Amacrine Cells
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For dye injections (Fig. 1 ), retinas were isolated from the eyecup, bisected and embedded in 2% agar-agar in Ringer's solution. Vertical sections (200 µm) were prepared as described previously (Dedek et al., 2006 (link)). AII amacrine cells were injected, under visual control, with microelectrodes (120–220 MΩ) filled with 7.5 mM Alexa-Fluor-594 potassium hydrazide (Invitrogen, Karlsruhe, Germany) diluted in 0.2 M KCl, pH 7.4. Cells were filled for 3–6 min. Tracer injections and whole-mount dye injections (Figs 4 –6 ) were performed as described previously (Dedek et al., 2006 (link); Shelley et al., 2006 (link)) using light-adapted mice. Retinal quarters were mounted, ganglion-cell side up, on black filter paper and incubated for 20 min in 0.1 mM DAPI diluted in Ringer's solution to visualize the nuclei of AII cells. Microelectrodes (120–180 MΩ resistance) were filled with 5 mM Alexa Fluor 488 and 4% (w/v) neurobiotin (Vector Laboratories, Burlingame, CA) or 7.5 mM Alexa Fluor 555 or 594. The Alexa dye was iontophoresed with 0.5 nA square pulses of 750 ms at 1 Hz for 1–2 min to visualize morphology; the current was reversed to inject positively charged neurobiotin (6–7 min), which was visualized with DyLight549-conjugated streptavidin (Jackson ImmunoResearch, West Grove, PA).
5
Dual Immunofluorescence Labeling of cl-C6
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To identify the protein expression of cl-C6, double-labelling immunofluorescence was performed using a cocktail of rabbit anti-cl-C6 (1:500) with a monoclonal antibody specific for either astrocytes (GFAP, 1:1,000; Sigma-Aldrich) or oligodendrocytes (Olig2, 1:2,000; Millipore, Temecula, Calif., USA) and a secondary antibody cocktail consisting of goat anti-mouse IgG conjugated to Alexa Fluor 488 (4 μg/ml; Invitrogen, Eugene, Oreg., USA) and goat anti-rabbit IgG conjugated to Alexa Fluor 546 (4 μg/ml; Invitrogen). Immunofluorescence was performed as previously described [30 (link)].
For double-labelling with cl-C6 and the microglia we stained cl-C6 with Alexa Fluor 546. The slides were then immersed in 4% paraformaldehyde for 5 min and incubated with biotinylated Lycopersicon esculentum (tomato) lectin (1:1,000; Vector Laboratories) and followed with streptavidin-conjugated Alexa Fluor 488 (Vector Laboratories).
For double-labelling with cl-C6 and the microglia we stained cl-C6 with Alexa Fluor 546. The slides were then immersed in 4% paraformaldehyde for 5 min and incubated with biotinylated Lycopersicon esculentum (tomato) lectin (1:1,000; Vector Laboratories) and followed with streptavidin-conjugated Alexa Fluor 488 (Vector Laboratories).
6
Visualizing Microglial Activation Markers
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Brain cryosections or treated BV2 cells were fixed with 4% paraformaldehyde (PFA) for 20 min, followed by permeabilization using a mixture of 0.5% Triton X‐100, 10% donkey serum, and 90% PBS for 60 min at room temperature. The samples underwent overnight incubation at 4°C with the following primary antibodies: anti‐Iba‐1 (1:500, rabbit, 019–19,741, WAKO, Japan), anti‐CD16 (1:500, goat, PA5‐47230, Thermo Fisher Scientific, USA), and anti‐CD206 (1:500, rat, MA5‐16871, Thermo Fisher Scientific, USA). After being washed with PBS, the brain slices/cells were incubated with secondary antibodies conjugated to either Alexa Fluor 488 or Alexa Fluor 546 (1:500, Vector Laboratories, USA) at room temperature for 60 min. Subsequently, the samples were washed three times with PBS, and then stained with 100 nM DAPI for 15 min. Immunofluorescent staining was observed and photographed using a fluorescence microscope (Olympus Corporation).
7
Brain Immunofluorescence Staining Protocol
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For brain immunofluorescence staining analysis, mice were anesthetized and transcardially perfused with 4% paraformaldehyde (PFA). The brains were dissected, postfixed in 4% PFA at 4 °C for 2 h, and then sectioned into 50-μm-thick slices by a VT1000S vibratome (Leica). The brain sections were pretreated in 0.5% Triton X-100 in phosphate-buffered saline (PBS, pH 7.4) for 1 h, blocked with 10% normal donkey serum, and 0.1% Triton X-100 in PBS for 1 h, and incubated with the indicated primary antibodies at 4 °C overnight. After washing with PBS, the samples were incubated with appropriate secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 546 (1:500, Vector Laboratories (Burlingame, CA, USA)) at room temperature for 1 h. The sections were counterstained with DAPI for 10 min, followed by washing in PBS. Images were acquired by ZEISS LSM880 confocal microscopy.
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