Lam-KD, LBR-KD or control S2 cells were seeded on coverslips for 30 min. After rinsing with PBS, cells were fixed in 100% methanol for 5 min at room temperature (for further examination of chromatin distribution based on the immunostaining of histone H4) or in 4% formaldehyde in PBS for 25 min at room temperature (for further estimation of chromatin volume based on DAPI staining), rinsed with PBS three times, blocked with PBTX (PBS with 0.1% Tween-20 and 0.3% Triton X-100) containing 3% normal goat serum (Invitrogen) for 1 h at room temperature. The remaining immunostaining procedure was performed as previously described50 (link). As primary antibodies we used murine monoclonal anti-histone H4 (1:200; ab31830, Abcam), guinea pig polyclonal anti-LBR26 (link) (1:1000), rabbit polyclonal anti-lamin Dm051 (link) (1:500). As the secondary antibodies we used Alexa Fluor 546-conjugated goat anti-rabbit IgG (Invitrogen) or Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen), or Alexa Fluor 633-conjugated goat anti-guinea pig IgG (Invitrogen).
Alexa fluor 633 conjugated goat anti guinea pig igg
Manufactured by Thermo Fisher Scientific
Alexa Fluor 633-conjugated goat anti-guinea pig IgG is a secondary antibody used for detection in immunoassays. It is conjugated to the Alexa Fluor 633 fluorescent dye, which can be excited using a red laser and emits light in the far-red region of the spectrum.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 546 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Ab31830, by Abcam (1 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Anti dig fitc, by Roche (1 mentions)
Dig dna labelling kit, by Roche (1 mentions)
Blocking solution, by Agilent Technologies (1 mentions)
Mouse anti brdu, by Agilent Technologies (1 mentions)
Rabbit anti glucagon, by Agilent Technologies (1 mentions)
Guinea pig anti insulin, by Agilent Technologies (1 mentions)
Fitc conjugated goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
4 protocols using alexa fluor 633 conjugated goat anti guinea pig igg
1
Immunostaining of Histone H4 and Chromatin
2
FISH Probe Generation and Hybridization
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~20-kb FISH probes were generated using a long-range PCR kit (Encyclo Plus PCR (Evrogen)) by PCR-amplification of 4 tiling genome fragments covering either the region 2 L:16964000–16982000 or 2 L:17310000–17328000, with the use of primer pairs provided in the Supplementary Table 1 . 1 µg of template DNA for hybridization was labelled by random primed synthesis with the DIG DNA labelling kit (Roche) or by ChromaTide Alexa Fluor 546–14-dUTP (Life Technologies). Probes were further combined and hybridized with S2 cells as described previously23 (link). For NL or FISH probe detection, as the primary antibodies we used guinea pig polyclonal anti-LBR26 (link) (1:1000), or rabbit polyclonal anti-lamin Dm051 (link) (1:500) and sheep polyclonal anti-DIG-FITC (1:500, Roche). As the secondary antibodies we used Alexa Fluor 633-conjugated goat anti-guinea pig IgG (Invitrogen), or Alexa Fluor 546-conjugated goat anti-rabbit IgG (Invitrogen) and Alexa Fluor 488-conjugated goat anti-FITC IgG (Invitrogen).
3
Immunohistochemistry of VGluT2 in Tissue
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All incubations were followed by washing with PBS-X. Sections were incubated successively with 10% normal goat serum (NGS) in PBS-X for 30 min, 1 µg/ml guinea pig anti-vesicular glutamate transporter type 2 (VGluT2) polyclonal antibody (Frontier Institute catalog #VGluT2-GP, RRID:AB_2571621; Miyazaki et al., 2003 (link)) in PBS-X containing 1% NGS and 0.02% sodium azide (PBS-XG) overnight, and Alexa Fluor 633-conjugated goat anti-guinea pig IgG (A-21105; Thermo Fisher Scientific) for 2 h. Sections were then mounted, counterstained, and coverslipped.
4
Immunohistochemical Analysis of Pancreatic Islets
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C57BL/6 mice were sacrificed at 4 weeks after rAd-BTC or rAd-βgal injection. Pancreata were removed, fixed in 10% formalin, and embedded in paraffin. More than 200 serial sections (4 μm thick) were prepared from each pancreas, and every 20–25th section was used for immunohistochemical analysis. The tissue sections were boiled (100°C for 10 min, 10 mM sodium citrate, pH 6.0) for antigen retrieval, and blocked with blocking solution (DAKO, Carpinteria, CA, USA). The sections were then incubated with primary antibody solution: guinea-pig anti-insulin (DAKO, 1:100), rabbit anti-glucagon (DAKO, 1:100), mouse anti-PDX-1 (DSHB, Iowa, IA, 1:100) or mouse anti-BrdU (DAKO, 1:50). Fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, 1:200), Texas Red (TR)-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology, 1:200) or Alexa-Fluor-633-conjugated goat anti-guinea-pig IgG (Thermo Fisher Scientific, Rockford, IL, 1:200) were used as secondary antibodies. Fluorescence was imaged using a laser scanning confocal fluorescent microscope (LSM 700, Carl Zeiss MicroImaging, Jena, Germany) and colocalization was analyzed using the ZEN 2009 Analysis Program.
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