Mab414

C. elegans embryos and larvae were collected and processed by freeze cracking and methanol fixation as described [40 (link)]. The following primary antibodies were used: mouse monoclonal antibody (mAb) 414 (Covance, Princeton, NJ, USA,1:250), mouse monoclonal antibody MH27 (1:50; [41 (link)], provided by the Developmental Studies Hybridoma Bank), rabbit polyclonal α-HCP-3 antiserum MH3N (1:200; generous gift from Dr. Mark Roth [42 (link)]), rabbit polyclonal α-NPP10-C/NUP96 antiserum GBLC (1:300; [21 (link)]), rabbit polyclonal α-MEL-28 antiserum BUD3 (1:200–250; [10 (link)]). Secondary antibodies were Alexa Fluor 546-conjugated goat anti-mouse antibodies (Invitrogen, 1:1000), Alexa Fluor 488- and Alexa Fluor 633-conjugated goat anti-rabbit antibodies (Invitrogen, 1:1000). For DNA staining, Hoechst 33258 (Hoechst) was used at 5 μg/ml. Confocal images for S1A Fig were obtained with a Nikon A1R microscope through a Plan Apo VC 60x/1.4 objective (Nikon, Tokyo, Japan) using a pinhole of 1 airy unit. All other immunofluorescence images were acquired with a confocal Leica SPE microscope equipped with an ACS APO 636/ 1.3 objective (Leica, Wetzlar, Germany) using a pinhole of 1 airy unit.

Sections on the wafers were washed with PBS (0.1 M, pH 7.4, 4 °C) for 20 min followed by incubation with 0.15% glycine in PBS (3 × 1 min) and washes in PBS (3 × 10 sec). Blocking solution composed of 0.5% BSA (Albumin Fraction V, Applichem) and 0.2% gelatin (from bovine skin, Sigma) in PBS was applied for 5 min. Primary antibodies, anti-NuP complex, (4 μg/ml, mAB414, Covance), anti-Tom20 (1 μg/ml, FL-145, Santa Cruz) and pNCC (1:250, antibody kindly provided by Prof. Johannes Loffing, University of Zurich, Switzerland) dissolved in blocking solution were incubated for 40 min at room temperature. After washes with blocking solution (6 × 20 sec) wafers were incubated with secondary antibodies (anti-mouse Alexa Fluor 568, 8 μg/ml, Life Technologies; anti-rabbit Alexa Fluor 488, 8 μg/ml, Life Technologies; anti-rabbit Alexa Fluor 647 F(ab’)₂, 6 μg/ml Jackson Immuno-Research for GSDIM experiments) in blocking solution (40 min at room temperature). For the immunogold experiments, 12 nm gold conjugated goat anti-rabbit antibodies were incubated for 40 min at room temperature (Optical density OD₅₂₅ 0.15). Washes in blocking solution (6 × 2 min) were followed by PBS (3 × 2 min) and post-fixation with 0.025% glutaraldehyde in PBS for 5 min. Finally, the wafers were transferred to a 6 well plate filled with PBS. Sections were stored at 4 °C until further processing.

Worms were transferred to a poly-l-lysine microscopy slide (VWR, Denmark), the cuticle was punctured with a sharp needle to allow for better exposure of the germline. A coverslip was gently placed on top and freeze-cracking was performed by incubation at −80 °C followed by removal of the coverslip from the frozen slides with a scalpel. The slides were incubated 20 min in ice cold methanol, washed in PBS, and then blocked 2 h in 2% (w/v) milk-PBS. Worms were encircled with a PAP pen (ThermoFischer, Denmark) before incubation with primary antibody against nuclear pore complex (mab414, Covance) 1:1000 dilution in 2% milk-PBS overnight. After incubation, the slides were washed 2 times in PBS and incubated 2 h with goat anti-mouse alexa546 conjugated antibody (ThermoFischer, Denmark) in 2% milk-PBS. The slides were washed 3 times in PBS, fixed 10 min in 2% PFA, and mounted with Vectashield antifade mounting media (Vector Laboratories). Confocal microscopy for co-localization of DLC-1::GFP and nuclear membrane was performed using a Zeiss LSM 780 microscope equipped with a Zeiss AxioCam MRm camera. The ZEN Imaging Software (Zeiss) was used to process the pictures.

The following antibodies were used: mouse monoclonal antibodies against α-tubulin (Santa Cruz, sc-23948), BAF (Abnova, H00008815-M07), GAPDH (Santa Cruz, sc-32233), PP2A (BD science, 610555), mAb414 (Covance, MMS-120P), Lamin A/C (Santa Cruz, sc-7292), GFP (Santa Cruz, sc-9996), phospho γ-H2A.X (Ser139) (EMD Millipore, 05-636) [all at a 1:500 dilution in phosphate-buffered saline (PBS) supplemented with 3% BSA] and Flag (Sigma-Aldrich, F1804) at a 1:5000 dilution in PBS supplemented with 3% BSA; and a rabbit polyclonal antibody against Lamin B1 (Abcam, ab16048) at a 1:500 dilution in PBS supplemented with 3% BSA. Horseradish peroxidase-conjugated anti-mouse (G21040) and anti-rabbit (G21234) antibodies were obtained from Invitrogen (used at a 1:5000 dilution in TBST). The following fluorochrome-conjugated secondary antibodies were used (at a 1:500 dilution in PBS supplemented with 3% BSA): anti-mouse Alexa Fluor-488 (Invitrogen, A11059), anti-rabbit Alexa Fluor-488 (Invitrogen, A11034), TRITC-conjugated phalloidin (Jackson Immunoresearch, P1951), anti-mouse Cy3 (Jackson Immunoresearch, 715-165-151), and Alexa Fluor-594 (Invitrogen, A11037).

For the immunostaining of gonads, worms were anesthetised in 0.001% tetramisole in M9 buffer. Gonads were dissected, snap frozen in liquid nitrogen and stained as described previously [25 (link)]. Slides were examined using an Olympus 1X81/1X2-UCB microscope. Primary antibodies used were: anti-PH3 (1/2000; rabbit) (Upstate Biotechnology), mAB414 (1/200; mouse) (Covance), MAPK (1/200; mouse) (Sigma). Secondary antibodies Alexa A488 and A555 (Invitrogen) were used at dilutions 1/1000 and 1/1200, respectively.
For live worm imaging, worms were anesthetized in a minimum of 0.001% tetramisole in M9 on a cover slip and mounted on a 1% agarose pad. Live worm slides were examined under the Olympus 1X81/1X2-UCB microscope or a Zeiss LSM 510 Meta confocal microscope. To observe sensory neurons in live RIOK::GFP transgenic worms, they were incubated with the lipophilic fluorescent dye Dil (Life Technologies) in the dark for three hours. Worms were then washed once with M9, plated and destained overnight and live worms were mounted on 1% agarose pads and examined by confocal microscopy. All images were processed using Adobe Photoshop and figures drawn using Adobe Illustrator.

Cells grown onto glass cover slips were fixed for 5 min with 4% PFA (SAV LP, Flinsbach, Germany) in PBS, permeabilised for 5 min with 0.5% Triton-X-100 in PBS and fixed again for 5 min. Blocking was performed for 30 min with 2% BSA/0.1% Triton-X-100 in PBS at room temperature.
All antibodies used for immunofluorescence were diluted in blocking solution. Primary antibodies anti-ALADIN (1:25), or anti-PGRMC2 (HPA041172; 1:50) or anti-PGRMC2 (F-3: sc-374624; 1:25) and anti-NPC proteins (mAb414; 1:800; Covance, Berkley CA, USA) were incubated at 4°C overnight in a humidified chamber. Secondary antibodies goat anti-mouse IgG Cy3 (1:800; Amersham Biosciences, Freiburg, Germany), Alexa Fluor 488 and 555 goat anti-rabbit IgG (1:500; Molecular Probes, Life Technologies) were incubated one hour at room temperature in the dark.
Fluorescence was visualised using the confocal laser microscope TCS SP2 (Leica Microsystems, Mannheim, Germany). The experiments were repeated at least three times.