Alexa fluor 568 goat anti mouse igg h l antibody

In the footpad spontaneous metastasis model, the popliteal lymph node was resected, fixed with 4% (w/v) PFA overnight at 4°C, and embedded in paraffin. Five-micron sections were deparaffinized and subjected to HE staining and immunofluorescence. For immunofluorescence, anti-CEA antibody (1:200 dilution, #10094, IBL) and anti-GFP antibody (1:1000 dilution, #04404-26, anti-GFP [Rat IgG2a] monoclonal [GF090R]; Nacalai Tesque, Tokyo, Japan) were used as primary antibodies. The sections were then washed three times in Tris-buffered saline with Tween 20 (TBS-T; 50 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.1% Tween 20), followed by incubation at room temperature for 3 h with Alexa Fluor 568-conjugated anti-mouse IgG (1:500 dilution, #A-11004, Alexa Fluor 568 Goat Anti-Mouse IgG [H+L] Antibody; Invitrogen) and Alexa Fluor 488-conjugated anti-mouse IgG (1:500 dilution, #A-11006, Alexa Fluor 488 Goat Anti-Rat IgG (H+L) Antibody; Invitrogen) as the secondary antibodies. Fluorescence and differential interference contrast images of the sections were observed with a wide-field fluorescence microscope (PlanApo 20×/0.75, PlanApo 40×/0.95, ECLIPSE 80i; Nikon).

Biopsy samples fixed in 10% buffered formalin were paraffin-embedded, sectioned at 4 µm, stained with Masson’s trichrome, periodic acid–Schiff, and hematoxylin–eosin–saffron, and examined by a pathologist that assessed Banff score.^{19 (link)}Immunofluorescence staining was performed on 8 µm sections of frozen tissue fixed in acetone. JM1E3 binding to the transplant was assessed by incubating the sections with an Alexa Fluor 568-goat anti-mouse IgG (H + L) antibody (Invitrogen, Carlsbad, CA) for 1 h at room temperature (RT). C5b-9 staining was performed with mouse IgG2 anti-human C5b-9 Ab (clone aE11, DIATEC, Norway) overnight at 4 °C followed by an Alexa Fluor 488–anti-mouse IgG2 Ab (Invitrogen) incubated for 1 h at room temperature (RT). For CD3 and CD68 staining, sections were incubated with Fab goat anti-mouse IgG (H + L) (Jackson ImmunoResearch) 2 h at RT, then incubated overnight at 4 °C with FITC-mouse anti-porcine CD3ε (clone PPT3, Southern Biotech, Birmingham, AL) or with mouse anti-rat CD68 (clone ED1, BIO-RAD) and finally with FITC-donkey anti-mouse IgG (H + L; Jackson ImmunoResearch). Sections were analyzed using a Nikon microscope and ACT-1 software (Nikon, Tokyo, Japan).

Localization of PALB2 mutants was visualized by immunofluorescence against the Flag-tag. HEK293T cells were fixed with 4% (w/v) paraformaldehyde for 15 min at room temperature, washed with TBS, and fixed again with ice-cold methanol for 5 min at −20°C. Cells were then permeabilized with 0.2% Triton X-100 in PBS for 5 min at room temperature. After three 5 min-washes in TBS, cells were quenched with 0.1% (w/v) sodium borohydride in PBS for 5 min. Cells were washed once with TBS and incubated for 45 min in blocking buffer (PBS, 10% (v/v) goat serum, 1% (w/v) BSA). Next, cells were incubated for 2 h at room temperature with the primary antibody (Monoclonal ANTI-FLAG^® M2 antibody produced in mouse, F3165, Sigma) diluted 1:500 in blocking buffer. After three 5 min-washes with TBS, cells were incubated for 45 min at room temperature in the dark with the secondary antibody (Alexa Fluor^® 568 Goat Anti-Mouse IgG (H+L) Antibody, A11004, Life Technologies) diluted 1:800 in TBS 1% (w/v) BSA. Coverslips were washed three times for 5 min with TBS and mounted onto slides using ProLong^® Gold Antifade Mountant (P36931, Life Technologies). The result was visualized using an epifluorescence microscope. RAD51 immunofluoresence was performed using 14B4 anti-RAD51 monoclonal antibody (Novus).