goat anti mouse igg2a jackson 115 005 206 cf568  (Biotium)

Journal: eLife

Article Title: A hierarchical pathway for assembly of the distal appendages that organize primary cilia

doi: 10.7554/eLife.85999

Figure Lengend Snippet: ( A ) and ( B ) 3D single-molecule data from showing FOP data from the two channels separately. The manually isolated localizations of CF568-labeled FOP (blue) and Alexa Fluor 647 (AF647)-labeled FOP (orange), shown for top and side views, along with CF568-labeled MYO5A (green) and AF647-labeled RAB34 (magenta) for the two samples. Orientations in the microscope 3D space are indicated by the inset axes. The opacities used for visualization in Vutara SRX are as follows: Sample 1, RAB34: 0.03, MYO5A: 0.08, FOP-CF568: 0.05, FOP-AF647: 0.05; sample 2, RAB34: 0.02, MYO5A: 0.09, FOP-CF568: 0.05, FOP-AF647: 0.05. ( C–F ) Example of reconstructions of the FOP ring structures in the two color channels after channel transformation and cross-correlation of FOP that is labeled in both channels. ( C–D ) Example of the AF647-labeled (magenta) and CF568-labeled (green) FOP structures at the mother and daughter centrioles used for cross-correlation. ( E–F ) The FOP structures from the two channels after channel transformation and data cross-correlation. The opacities for the visualization are set in Vutara SRX to 0.07 for AF647 FOP and 0.02 for CF568 FOP.

Article Snippet: In the experiments shown in , the cells were incubated with rabbit anti-RAB34 (27435–1-AP, Proteintech, 1:500), mouse IgG2a anti-RAB34 (sc-365986, Santa Cruz, 1:250), and mouse IgG2b anti-FOP (H00011116-M01, Abnova, 1:1000) diluted in 1% BSA in PBS at 4 °C overnight, washed three times in 0.1% Triton-X 100 in PBS, and then incubated with donkey anti-rabbit Alexa Fluor 647 (ab150067, Abcam, 1:1000), goat anti-mouse IgG2b Alexa Fluor 647 (A-21242, Invitrogen), goat anti-mouse IgG2a CF568 (20258, Biotium), and goat anti-mouse IgG2b CF568 (20268, Biotium) diluted in 1% BSA in PBS for 1 hr shielded from light.

Techniques: Isolation, Labeling, Microscopy, Transformation Assay

Journal: eLife

Article Title: A hierarchical pathway for assembly of the distal appendages that organize primary cilia

doi: 10.7554/eLife.85999

Figure Lengend Snippet: ( A ) Super-resolution reconstructions from the localizations of CF568-labeled RAB34 and Alexa Fluor 647 (AF647)-labeled RAB34. The double-sided arrows specify the axes of the histograms shown in ( B ). ( B ) Offsets between the center of masses of CF568-labeled RAB34 and AF647-labeled RAB34 were found by fitting normalized histograms along each axis to Gaussian functions and calculating the difference between the two peaks. This was done in the x, y, and z directions before finding the total offset in 3D space. The offset between the localizations was found to be 20 nm in this sample, and there were 1705 localizations in the red channel and 1228 localizations in the green channel for RAB34. ( C ) Super-resolution reconstruction of RAB34 and FOP in both channels shown for top view (top) and side view (bottom) after channel registration. Visualizations were rendered in Vutara SRX as 3D Gaussians with 20 nm diameter. The opacities used for visualization are as follows: ( A ) RAB34-Alexa647: 0.06, RAB34-CF568: 0.06; ( C ) RAB34-AF647: 0.1, RAB34-CF568: 0.1, FOP-AF647: 0.05, FOP-CF568: 0.05.

Article Snippet: In the experiments shown in , the cells were incubated with rabbit anti-RAB34 (27435–1-AP, Proteintech, 1:500), mouse IgG2a anti-RAB34 (sc-365986, Santa Cruz, 1:250), and mouse IgG2b anti-FOP (H00011116-M01, Abnova, 1:1000) diluted in 1% BSA in PBS at 4 °C overnight, washed three times in 0.1% Triton-X 100 in PBS, and then incubated with donkey anti-rabbit Alexa Fluor 647 (ab150067, Abcam, 1:1000), goat anti-mouse IgG2b Alexa Fluor 647 (A-21242, Invitrogen), goat anti-mouse IgG2a CF568 (20258, Biotium), and goat anti-mouse IgG2b CF568 (20268, Biotium) diluted in 1% BSA in PBS for 1 hr shielded from light.

Techniques: Labeling

Journal: eLife

Article Title: A hierarchical pathway for assembly of the distal appendages that organize primary cilia

doi: 10.7554/eLife.85999

Figure Lengend Snippet: Single fluorophores of CF568 and AF647 are excited by 560 nm and 642 nm lasers, respectively, using widefield epi-illumination. A 100 x objective lens serves for both illumination and collection of emitted light. A 4 f optical relay system is used to image the emitted light, which is split by a dichroic mirror into two emission paths. Transmissive phase masks in the Fourier plane of both paths modulate the emission light, thereby changing the shape of the point spread function (PSF) to that of the double helix PSF, which encodes the 3D position of the emitter. The two paths which are split in the 4 f system are imaged onto separate regions of an EMCCD camera. FOP is labeled with both dyes and is imaged in both channels, whereas MYO5A and RAB34 are labeled and imaged in channels 1 and 2, respectively, as indicated above. The inset shows the shape of the double helix PSF in both channels at different axial ( z ) positions. Scale bar is 1 µm. The schematic is not drawn to scale.

Article Snippet: In the experiments shown in , the cells were incubated with rabbit anti-RAB34 (27435–1-AP, Proteintech, 1:500), mouse IgG2a anti-RAB34 (sc-365986, Santa Cruz, 1:250), and mouse IgG2b anti-FOP (H00011116-M01, Abnova, 1:1000) diluted in 1% BSA in PBS at 4 °C overnight, washed three times in 0.1% Triton-X 100 in PBS, and then incubated with donkey anti-rabbit Alexa Fluor 647 (ab150067, Abcam, 1:1000), goat anti-mouse IgG2b Alexa Fluor 647 (A-21242, Invitrogen), goat anti-mouse IgG2a CF568 (20258, Biotium), and goat anti-mouse IgG2b CF568 (20268, Biotium) diluted in 1% BSA in PBS for 1 hr shielded from light.

Techniques: Labeling