Neonatal and post-neonatal (0–3 weeks old) intestine was fixed in 10% formalin and embedded in paraffin, whereas post-weanling and adult intestine were fixed and embedded as described previously27 (link). Briefly, tissue was fixed in 1% paraformaldehyde, washed with 50 mM NH4Cl, cryoprotected in 30% sucrose (wt/vol) and embedded in Optimal Cutting Temperature (OCT, Tissue-Tek) medium. Immunostaining of frozen or FFPE sections (5–7 μm) was performed using the rabbit anti-laminin (Sigma-Aldrich) or biotin-labeled anti-GFP (Abcam), followed by Alexa Fluor 594 goat anti-rabbit IgG (H+L), Alexa Fluor 647 phalloidin, Alexa Fluor 647 Streptavidin (Invitrogen) and/or Hoechst 33342 dye (Invitrogen). Slides were mounted with ProLong Glass (Invitrogen) and images were acquired on an inverted DMi8 microscope (Leica) equipped with a CSU-W1 spinning disk, ZYLA SL150 sCMOS camera (Andor), PL APO 40x/0.85 dry objectives, and iQ3 acquisition software (Andor). The number of GFP+ cells per 0.1 mm2 villus was quantified by an observer blinded to the condition.
Csu w1 spinning disk
Manufactured by Oxford Instruments
The CSU-W1 is a spinning disk confocal microscope system. It utilizes a spinning Nipkow disk to generate multiple excitation and detection pinholes, enabling parallel confocal imaging. The system provides high-speed optical sectioning and efficient light utilization for live-cell imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti laminin, by Merck Group (2 mentions)
Prolong glass, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342 dye, by Thermo Fisher Scientific (2 mentions)
Biotin labeled anti gfp, by Abcam (2 mentions)
Streptavidin alexa fluor 647, by Thermo Fisher Scientific (2 mentions)
Zyla sl150 scmos camera, by Oxford Instruments (2 mentions)
Iq3 acquisition software, by Oxford Instruments (2 mentions)
Dmi8 microscope, by Leica (2 mentions)
Alexa fluor 594 goat anti rabbit igg h l, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (2 mentions)
μ slide 18 well, by Ibidi (1 mentions)
Anti icp0, by Santa Cruz Biotechnology (1 mentions)
3 protocols using csu w1 spinning disk
1
Immunostaining of Intestinal Tissue Sections
2
SARS-CoV-2 and Herpes Virus Immunofluorescence
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Cells were pelleted at 16,000 xg for 1 min and resuspended in 50 μL PBS. Cells were added to a μ-Slide 18 well (Ibidi, # 81826) for 2 s. Slides were allowed to dry for 30 min at room temperature before inactivation with 100% acetone for 30 min at room temperature. SARS2-CoV-2 samples were blocked with 1% BSA in PBS, stained with anti-N hybridoma 5D4 supernatant diluted 1:10 in 1% BSA in PBS, and stained with anti-mouse AF488 diluted 1:500 and DAPI at 1 ug/mL. The SARS2-CoV-2 antibody was a kind gift from Petra Emmerich. PBS with 1% FBS 0.1% Triton X-100 was used to block HSV-1 and HCMV samples and dilute the antibodies. Anti-ICP0 (Santa Cruz Biotechnology, # sc-53070) diluted 1:100 was used for HSV-1, and anti-IE1/2 hybridoma 3H4 supernatant diluted 1:3 was used for HCMV.
Images were acquired with a Nikon Eclipse Ti2 body equipped with a Yokogawa CSU-W1 spinning disk, Andor iXon Ultra DU-888U3 EMCCD camera, and Plan Apo 20x objective. Pixel resolution was 655 nm/pixel. Illumination was performed with a 405 nm and 488 nm laser through a quad filter (405/488/568/647), and emission light was acquired with a 447/60 and 525/50 filter for DAPI and AF488, respectively.
Images were acquired with a Nikon Eclipse Ti2 body equipped with a Yokogawa CSU-W1 spinning disk, Andor iXon Ultra DU-888U3 EMCCD camera, and Plan Apo 20x objective. Pixel resolution was 655 nm/pixel. Illumination was performed with a 405 nm and 488 nm laser through a quad filter (405/488/568/647), and emission light was acquired with a 447/60 and 525/50 filter for DAPI and AF488, respectively.
3
Immunostaining of Intestinal Tissue Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Neonatal and post-neonatal (0–3 weeks old) intestine was fixed in 10% formalin and embedded in paraffin, whereas post-weanling and adult intestine were fixed and embedded as described previously27 (link). Briefly, tissue was fixed in 1% paraformaldehyde, washed with 50 mM NH4Cl, cryoprotected in 30% sucrose (wt/vol) and embedded in Optimal Cutting Temperature (OCT, Tissue-Tek) medium. Immunostaining of frozen or FFPE sections (5–7 μm) was performed using the rabbit anti-laminin (Sigma-Aldrich) or biotin-labeled anti-GFP (Abcam), followed by Alexa Fluor 594 goat anti-rabbit IgG (H+L), Alexa Fluor 647 phalloidin, Alexa Fluor 647 Streptavidin (Invitrogen) and/or Hoechst 33342 dye (Invitrogen). Slides were mounted with ProLong Glass (Invitrogen) and images were acquired on an inverted DMi8 microscope (Leica) equipped with a CSU-W1 spinning disk, ZYLA SL150 sCMOS camera (Andor), PL APO 40x/0.85 dry objectives, and iQ3 acquisition software (Andor). The number of GFP+ cells per 0.1 mm2 villus was quantified by an observer blinded to the condition.
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