THP-1 cells were cultured as described above and seeded at 1 × 105 cells per 13 mm coverslip and differentiated with PMA as described above. GFP-expressing M. bovis BCG was incubated with 0, 1, 10 µg/ml of TSR4+5 for 2 h at 37°C in buffer 1. Cells were also incubated with 10 µg/ml of BSA as a negative control. Cells were washed twice in PBS and then resuspended in plain RPMI media. 1 × 106 GFP-M. bovis BCG was added to the THP-1 cells (MOI of 10:1) and incubated for phagocytosis for 2 h at 37°C. THP-1 cells were then washed three times in PBS to remove extracellular bacteria and then fixed in 4% paraformaldehyde for 5 min. After washing three times in PBS, THP-1 cells were incubated with 2 µg/ml of AlexaFluor546-conjugated wheat germ agglutinin (Invitrogen) to reveal the plasma membrane. Cells were then washed three times and mounted using Vectashield antifade with DAPI (Vector Labs) to reveal nucleus. Slides were observed under a Leica DM4000 fluorescence microscope at 40× magnification. Images were processed using Image J (see text footnote 1).
Dm4000 fluorescence microscope
Manufactured by Leica camera
Sourced in Germany
The Leica DM4000 is a fluorescence microscope designed for advanced imaging and analysis. It features high-resolution optics and a range of illumination options to enable detailed observation and imaging of fluorescently-labeled samples. The core function of the DM4000 is to provide a versatile platform for fluorescence microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Dapi containing mounting medium, by Vector Laboratories (2 mentions)
Vectashield antifade with dapi, by Vector Laboratories (1 mentions)
Hrp donkey anti goat igg, by Proteintech (1 mentions)
Live dead kit, by Thermo Fisher Scientific (1 mentions)
Syto9, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse fitc, by Proteintech (1 mentions)
Goat anti rabbit cy3, by Proteintech (1 mentions)
4 6 diamidin 2 phenylindol dapi, by Merck Group (1 mentions)
Phalloidin tetramethylrhodamine b isothiocyanate, by Merck Group (1 mentions)
Mem 108, by BioLegend (1 mentions)
Um7f8, by BD (1 mentions)
Dylight 488, by Jackson ImmunoResearch (1 mentions)
Glutamine, by Merck Group (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
9 protocols using dm4000 fluorescence microscope
1
Phagocytosis of M. bovis BCG by THP-1 cells
2
Immunohistochemical Analysis of IL-1β
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The deparaffinized sections were subjected to heat mediated antigen retrieval, and blocked in H2O2 for 30 minutes, followed by PBS with 5% BSA and 0.2% Triton X‐100 at room temperature for 30 minutes, and then stained overnight with primary antibody against IL‐1β (R&D System, Cat#AF‐401‐NA). After rinsing with PBS for 15 minutes, tissues were incubated with secondary antibody (HRP‐Donkey Anti‐Goat IgG, Proteintech, Cat#SA00001‐3) at room temperature. Sections were then washed and colours were developed with DAB (3,3’‐diaminobenzidine). Next, haematoxylin was used as a counterstain. Images were captured with a Leica DM4000 fluorescence microscope.
3
Histological Analysis of Maxillae
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolated maxillae were fixed in 4% paraformaldehyde, and then decalcified in 14% EDTA. After dehydration, the maxillae were embedded in paraffin for paraffin sections or embedded in Tissue‐Tek OCT compound for frozen sections. The paraffin sections were stained with haematoxylin and eosin (H&E), histochemically for tartrate‐resistant acid phosphatase (TRAP) or alkaline phosphatase (ALP) and analysed as described previously.18 The frozen sections of Lysm‐Cre/RosanTnG mice were mounted with Mounting Medium containing DAPI (Vector) and images were captured with a Leica DM4000 fluorescence microscope, as we reported previously.19
4
Cell Death Visualization via Confocal Microscopy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cell death was assessed by using confocal microscopy. The cells were stained using the Live/Dead Kit containing PI and SYTO 9 (Invitrogen). Cells grown to an OD600 of 0.8 were washed twice with 0.85% NaCl. Cells treated with a sub-lethal dose of MMC (15 μg/ml, 40 min), as well as untreated cells, were collected by centrifugation. Then the cells were stained using 1 μl of a 1:1 mixture of PI and SYTO 9 from the kit and were incubated for 15 min at room temperature keeping in the dark. Following the wash by 0.85% NaCl twice, 100 μl of 4% formalin was applied for the fixation. The cells were washed twice with PBS buffer and re-suspended in 20 μl of 50% glycerol. A confocal microscopy assay was performed using a Leica DM4000 fluorescence microscope. Living cells were stained by SYTO 9 (green) and death cells were stained by PI (red).
5
FJC Staining of Retinal Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
FJC staining (MilliporeSigma, Burlington, MA, USA) was performed on 20-μm thick retinal sections according to manufacturer's instructions, with slight modifications. Briefly, sections were immersed in a basic alcohol solution (1% NaOH in 80% EtOH) for 5 minutes, followed by 2 minute rinses in 70% EtOH and distilled water each. Blocking was performed with a 10-minute incubation in 0.06% potassium permanganate solution, followed by a 2-minute rinse in distilled water. A FJC working solution (0.001%) containing Hoechst (Life Technologies) was placed onto retinal sections and incubated at room temperature for 10 minutes. Slides were washed three times in distilled water and dried at 50°C on a slide warmer. Xylene was used before mounting in DPX nonfluorescent mounting media (MilliporeSigma). Staining was visualized using a Leica DM4000 fluorescence microscope.
6
Immunohistochemical and Immunofluorescence Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The deparaffinized sections were subjected to heat mediated antigen retrieval, and blocked in H2O2 for 30 minutes, followed by PBS with 5% BSA and 0.2% Triton X-100 at room temperature for 30 minutes, and then stained overnight with primary antibody against NLRP3 (Abcam, Cat#ab214185), Prx1 (Origene, Cat#TA803116), USP1 (CST, Cat#D37B4) or UCHL5 (Abcam, Cat#ab133508) at 4 °C. For immunohistochemistry staining, after rinsing with PBS for 15 minutes, tissues were incubated with HRP-conjugated secondary antibody at room temperature. Sections were then washed and colors were developed with DAB (3,3ʹ-diaminobenzidine). Next, hematoxylin was used as a counterstain. For immunofluorescence staining, tissues were incubated with goat anti-mouse FITC (Proteintech, Cat#SA00003-1) or goat anti-rabbit Cy3 (Proteintech, Cat#SA00009-2) at room temperature. Slides were mounted with Mounting Medium containing DAPI (Vector Labs, Burlingame, CA, USA). All the images were captured with a Leica DM4000 fluorescence microscope.
7
Immunofluorescence Staining of Retinal Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single and double immunofluorescence staining were performed on paraffin-embedded retinal sections processed as described in Section 2.4 . and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) according to the protocol described in Table 4 . Table 5 lists used primary and secondary antibodies. After mounting, micrographs were recorded at 100×, 200×, and 400× magnification using the Leica DM4000 fluorescence microscope, taking three images from every sample (right side, left side, center).
8
Indirect Immunofluorescence Staining Protocol
9
Nanoparticle Interaction with Lymphocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In order to investigate the interaction of nanoparticles with lymphocytes by flow cytometry, nanoparticles were fluorescentlabeled by the addition of 2% of Egg Liss Rhod PE (L-a-Phosphatidylethanolamine-N-(lissaminerhodamine B sulfonyl)) in relation to the main structural components of each nanoparticle. Peripheral human blood was obtained by venipuncture from healthy adult donors, diluted with an equal volume of RPMI 1640 medium, then layered over Ficoll-Hypaque density gradient separation solution (1.077 g/ml), and centrifuged at 400g for 20 min at room temperature. The mononuclear cell layer was removed, washed twice in RPMI 1640 medium and resuspended in RPMI 1640 medium supplemented with 2 mM glutamine (Sigma Chemical Co.), antibiotics and 10% FCS then the cells were cultured with 10 lg/mL of phytohemagglutinin. Lymphocytes were incubated with 2.1 Â 10 9 nanoparticles for up to 6 h. After incubation, as described above, the lymphocytes were incubated and the analysis (10.000 events were collected per sample) was performed with a FACS Canto II flow cytometer (Becton Dickinson, California, USA) using the FACS Diva software. In addition, the lymphocytes were directly observed in a Leica DM4000 fluorescence microscope (Wetzlar, Germany).
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!