Kidneys frozen in liquid nitrogen were embedded in Optimal Cutting Temperature compound (Ted Pella Inc.). Tissue was cryosectioned (5 μm) and permeabilized with cold acetone for 20 min at room temperature. The slides were washed with PBS and blocked with 2% horse serum prepared in 3% BSA/PBS for 20 min at room temperature. The sections were stained for 30 min with 1:200 diluted horse anti-mouse IgG-Fluorescein (Vector Laboratories) followed by extensive washing in PBS containing 0.1% Tween 20 and mounting using ProLong Diamond Antifade (Thermo Fisher Scientific) containing DAPI counterstain. Images were acquired using Axiovert 200 microscopy system with Apotome imaging and Axiovision software (Carl Zeiss Microscopy, LLC). For GC studies, spleen sections were rehydrated for 30 min with PBS and blocked for 1 h with 10% BSA/PBS at room temperature. The sections were stained for 1 h with 1:100 diluted PNA-Fluorescein (Vector Laboratories) or anti-GL7-Alexafluor 488 and washed three times with PBS, followed by incubation with 1:50 diluted PE-anti-mouse CD19 antibody (Thermo Fisher Scientific) at room temperature for 1 h. The slides were washed and mounted using ProLong Diamond Antifade (Thermo Fisher Scientific) containing DAPI. Germinal centers were mapped using the selection tool and quantified using ImageJ (NIH). H & E stained spleen sections were also imaged for GC analyses.
Prolong diamond antifade
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
ProLong Diamond Antifade is a mounting medium designed for fluorescence microscopy. It is formulated to reduce photobleaching and maintain the brightness of fluorescent signals in fixed samples.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (15 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (14 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (6 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (6 mentions)
Lsm 780 confocal microscope, by Zeiss (6 mentions)
Lsm 710 confocal microscope, by Zeiss (6 mentions)
Sp8 confocal microscope, by Leica (5 mentions)
Cryostat, by Leica (4 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (4 mentions)
Lsm 880 confocal microscope, by Zeiss (4 mentions)
Lsm 880, by Zeiss (4 mentions)
Alexa fluor 568 phalloidin, by Thermo Fisher Scientific (3 mentions)
Zen software, by Zeiss (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
Inform software, by PerkinElmer (3 mentions)
Halo software, by Indica Labs (3 mentions)
Laser scanning confocal microscope lsm710, by Zeiss (3 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
164 protocols using prolong diamond antifade
1
Detailed Immunofluorescence Kidney and Spleen Analysis
2
Quantitative Immunofluorescence Imaging of Kidney and Spleen
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Kidneys frozen in liquid nitrogen were embedded in Optimal Cutting Temperature compound (Ted Pella Inc.). Tissue was cryosectioned (5 μm) and permeabilized with cold acetone for 20 min at room temperature. The slides were washed with PBS and blocked with 2% horse serum prepared in 3% BSA/PBS for 20 min at room temperature. The sections were stained for 30 min with 1:200 diluted horse anti‐mouse IgG‐Fluorescein (Vector Laboratories) followed by extensive washing in PBS containing 0.1% Tween 20 and mounting using ProLong Diamond Antifade (Thermo Fisher Scientific) containing DAPI counterstain. Images were acquired using Axiovert 200 microscopy system with Apotome imaging and Axiovision software (Carl Zeiss Microscopy, LLC). For GC studies, spleen sections were rehydrated for 30 min with PBS and blocked for 1 h with 10% BSA/PBS at room temperature. The sections were stained for 1 h with 1:100 diluted PNA‐Fluorescein (Vector Laboratories) or anti‐GL7‐Alexafluor 488 and washed three times with PBS, followed by incubation with 1:50 diluted PE‐anti‐mouse CD19 antibody (Thermo Fisher Scientific) at room temperature for 1 h. The slides were washed and mounted using ProLong Diamond Antifade (Thermo Fisher Scientific) containing DAPI. Germinal centers were mapped using the selection tool and quantified using ImageJ (NIH). H & E stained spleen sections were also imaged for GC analyses.
3
Immunostaining and Microscopy Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples were prepared by first seeding 106 cells onto a 15 mm #1.5 round glass coverslip coated with poly-L-lysine (Sigma-Aldrich). Following seeding, the samples were fixed by incubating in PBS containing 4% paraformaldehyde for 30 min at room temperature, then permeabilized using 0.1% TX-100 in PBS with 10 mM glycine (PBS-glycine). For immunostaining, fixed and permeabilized cells were incubated with 0.5–1.0 μg of primary antibody in 500 μl of PBS-glycine at room temperature for 45 min, followed by washing and incubation with 2.0 μg of secondary antibody for 45 min at room temperature. Following immunostaining, the samples were labeled with DAPI using a 300 nM solution in PBS-glycine for 5 min at room temperature. The samples were mounted using ProLong Diamond Antifade (Thermo Fisher, Waltham, MA).
Confocal microscopy was performed using a Zeiss LSM-710 equipped with a 63x 1.4 NA objective (OMRF Imaging Core). Structured Illumination Microscopy (SIM) was performed using DeltaVision OMX-SR system (OMRF Imaging Core).
Confocal microscopy was performed using a Zeiss LSM-710 equipped with a 63x 1.4 NA objective (OMRF Imaging Core). Structured Illumination Microscopy (SIM) was performed using DeltaVision OMX-SR system (OMRF Imaging Core).
4
Immunohistochemical Analysis of Brain Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Animals were deeply anesthetized with Fatal Plus (Vortech Pharmaceuticals) and transcardially perfused with PBS, 0.1 m , pH 7.4, followed by 4% paraformaldehyde (PFA; 4% in PBS), pH 7.2. Brains were removed and postfixed for 24 h in 4% PFA at 4°C, then kept in 30% sucrose at 4°C until fully submerged. Coronal sections (45 μm thick) were cut on a cryostat (model CM2050S, Leica) and directly mounted onto slides. Slide-mounted sections were washed in PBS, blocked with 5% normal goat serum (NGS) in PBS + 0.4% Triton X-100 (PBS-T) for 1 h, and incubated with anti-GFP (1:1000; catalog #ab13970, Abcam) and anti-Iba1 (1:1000; catalog #019–19 741, Wako) in 2% NGS in PBS-T overnight. The next day, slides were incubated with Alexa Fluor 488 (1:500; Thermo Fisher Scientific) and Alexa Fluor 594 (1:500; Thermo Fisher Scientific) in PBS-T for 2 h, washed and stained with Hoechst 33342 (1:3000; catalog #H3570, Thermo Fisher Scientific) for 10 min, and coverslipped with ProLong Diamond Antifade (Thermo Fisher Scientific).
5
Brain Tissue Collection and Immunohistochemistry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were injected with euthanasia solution and subsequently sacrificed via transcardial perfusion, first with PBS and then with paraformaldehyde (PFA; 4% in PBS). Brains were then postfixed in 4% PFA at 4 °C overnight, and cryoprotected using a 30% PBS-buffered sucrose solution for ~24–36 h. Coronal brain sections (45 μm) were acquired using a freezing microtome (SM 2010R, Leica). For immunostainings, brain sections were blocked in 10% normal goat serum (NGS) in PBST (0.1% Triton X-100 in PBS) for 1 h at RT, followed by incubation with primary antibodies in 10% NGS-PBST for 48 h at 4 °C. Sections were then washed with PBST (3 × 15 min) and incubated with fluorescent secondary antibodies at RT for 1 h in 10% NGS-PBST. Sections were washed in PBS (3 × 15 min), mounted onto glass slides and coverslipped with ProLong Diamond antifade (ThermoFisher Scientific). Images were taken using a Carl Zeiss LSM 780 confocal microscope using ZEN (version 2.3, Carl Zeiss Microscopy, LLC). Image analysis and cell counting were performed using ImageJ software by a blinded experimenter (Fiji, version 2017 May 30). Optical fiber placements for all subjects included in this study are presented in Supplementary Fig. 11 .
6
Immunostaining of Cytoskeletal Components
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed with 4% paraformaldehyde (PFA) (Electron Microscopy Sciences) for 10 min at 37 °C. PFA was quenched with 0.1 M glycine and cells were permeabilized with 0.1% Triton X-100, both for 10 min at room temperature. The actin cytoskeleton was stained with Alexa Fluor-conjugated phalloidin (Alexa Fluor 568-Phalloidin or Alexa Fluor 647-Phalloidin; Thermo Fisher) diluted to 2.5% in phosphate-buffered saline (PBS); nuclei were stained with Hoechst 33342 (Thermo Scientific); pMLC was immunolabeled using human anti-(Thr18/Ser19) pMLC 2 no. 3674 (Cell Signaling) at 1:100 dilution; Arp2/3 was immunolabeled using human anti-Arp2/3 clone no.13C9 (Millipore) at 1:200 dilution; EVL was immunolabeled using human anti-EVL (a gift from Frank Gertler) at 1:50 dilution. Slides were blocked in 1% FBS/1% bovine serum albumin (BSA) in PBS; primary and secondary antibodies were diluted in blocking buffer and incubated using standard protocols. Samples were mounted in ProLong Diamond Antifade (Thermo Fisher) and allowed to cure for at least 24 h before imaging.
7
Enumeration of Intracellular Bacterial Communities
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IBC enumeration was performed as described previously using bacterial strains transformed with pCOM::GFP plasmid (13 (link)). Mice were euthanized 6 hpi and the bladders were removed with aseptic technique. Mouse bladders were stretched, pinned, and fixed with 3.4% paraformaldehyde overnight at 4 °C. Bladders were washed twice in 1X PBS, permeabilized with 0.1% Triton X-100 for 15 min, followed by a 1XPBS wash. Bladders were stained at room temperature with Alexa Fluor 568 Phalloidin (ThermoFisher) and mounted on to slides with ProLong Diamond Antifade (ThermoFisher). IBCs were manually counted via fluorescence microscopy on a LSM 710 confocal laser scanning microscope (Zeiss).
8
Myotube Differentiation Modulated by Cytokines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Myotubes were differentiated for 8 d in 24-well culture plates in the presence of rIL-15 (0, 1, 25, 100 ng/mL), rTNFα (0, 1 ng/mL) or both cytokines (rIL-15 25 ng/mL and rTNFα 1 ng/mL). Media were renewed every 2 d. The culture medium was removed and the cells fixed with 2% formaldehyde in PBS for 30 min. Following permeabilization in 100% methanol for 10 min, wells were blocked with 5% goat serum in PBS for 30 min. Primary antibodies were diluted (Desmin, 1:1000, Dako) in 1% BSA/PBS and 150 μL was added per well for 1 hour. Wells were subsequently incubated with 150 μL/well secondary antibody (Goat anti-Mouse IgG (H + L), Alexa Fluor® 488 conjugated, Thermo Fisher) for 1 hour in the dark. Each well was washed with PBS and 150 μL/well DAPI/PBS (1:5000, Cell Signalling Technology) was added for 5 min in the dark. Wells were further washed with PBS, a drop of mountant added to each well (ProLong Diamond Antifade, Thermo Fisher) and a coverslip applied.
9
Immunofluorescent Analysis of Spleens in Bone Marrow Chimera Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spleens from bone marrow chimera mice were embedded in optimal cutting temperature compound and snap frozen in liquid nitrogen. 4-μm cryosections were briefly fixed in 4% neutral buffered formalin and stained with anti-14E06, anti-CD169/SIGLEC (clone 3D6.112; BioRad), and anti-IgM (donkey-anti-mouse-IgM-A488; Jackson ImmunoResearch) followed by donkey-anti-rat-IgG-Cy3,donkey-anti-human-IgG-biotin(both Jackson ImmunoResearch), and streptavidin-APC (ProZyme), or anti-14E06, anti-B220-biotin (clone RA3-6B2; BioLegend), and anti-CD3-PE (clone 145-2C11; BioLegend) followed by donkey-anti-human-IgG-A488 and streptavidin–aminomethylcoumarin acetate (both Jackson ImmunoResearch). Slides were mounted with ProLong Diamond Antifade (Thermo Fisher Scientific). Images were acquired on an inverted fluorescence microscope (Nikon Eclipse Ti-S; Nikon) with a Nikon 9 10/0.3 or 9 20/0.45 Plan Fluor lens (Nikon) and NIS ELEMENTS BR3.2 software (Nikon). Single channel images were merged in FIJI/ImageJ (Schindelin et al., 2012 (link); Schneider et al., 2012 (link)). Linear adjustment of brightness and contrast was done equally across images. Anti-14E06 staining acquired in the blue channel was pseudocolored as green and anti-IgM staining acquired in the green channel was pseudocolored as blue for visualization purposes.
10
Immunohistochemical Analysis of Mammary Tissue
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!