The lung sections were washed with PBS, and the samples were blocked in antibody dilution buffer of 2% BSA/PBS for 15 min at room temperature (RT). After removal of the blocking solution, primary antibodies/markers were added to antibody dilution buffer at 37 °C for 2 h: isolectin B4 (ILB4) (1:100) (VECTOR Laboratories, Burlingame, CA) for endothelial cells; anti-CD3 (1:200) (eBioscience, San Diego, CA, USA) for T lymphocytes; anti-F4/80 antibody (1:200) (eBioscience) for macrophages; and Gr-1 (1:200) (eBioscience) for neutrocytes. After washing with PBS, cells were incubated for 30 min at RT with secondary antibodies prepared at 1:500 in antibody dilution buffer: Alexa 488 goat anti-rat IgG and Alexa 594 goat anti-rat IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). After the secondary antibodies were removed and the tissues were washed with PBS, nuclear counterstaining was performed by incubation with 4′,6-diamidino-2-phenylindole (DAPI) solution (1 µg/mL in PBS; Sigma-Aldrich Japan K.K.) for 10 min at RT. The sample slides were covered by cover slips with mounting medium (ImmunoBioScience, Mukilteo, WA) followed by sealing with nail varnish before evaluation under a fluorescence microscope (BZx-700, Keyence, Osaka, Japan). The positively stained cells in each sample were counted in 5 different high power fields (×200).
4 6 diamidino 2 phenylindole dapi solution
Manufactured by Merck Group
Sourced in United States, Japan
4′,6-diamidino-2-phenylindole (DAPI) solution is a fluorescent dye used in molecular biology and microscopy applications. It selectively binds to DNA, allowing visualization and detection of nucleic acids in cells and tissues. DAPI is a common and widely used stain for fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (9 mentions)
Zen software, by Zeiss (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Prolong glass, by Thermo Fisher Scientific (2 mentions)
Cm1950, by Leica camera (2 mentions)
Leica tcs sp8 confocal laser scanning microscope, by Leica camera (2 mentions)
Image pro plus 6, by Media Cybernetics (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Isolectin b4 ilb4, by Vector Laboratories (1 mentions)
Alexa 488 goat anti rat igg, by Jackson ImmunoResearch (1 mentions)
Anti f4 80 antibody, by Thermo Fisher Scientific (1 mentions)
Anti cd3, by Thermo Fisher Scientific (1 mentions)
Bz x700, by Keyence (1 mentions)
Ab24345, by Abcam (1 mentions)
Sp5 2 confocal microscope, by Leica camera (1 mentions)
A11035, by Thermo Fisher Scientific (1 mentions)
Anti acetylated α tubulin antibody, by Cell Signaling Technology (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
A28175, by Thermo Fisher Scientific (1 mentions)
33 protocols using 4 6 diamidino 2 phenylindole dapi solution
1
Lung Tissue Immunofluorescence Staining
2
Immunohistochemical Analysis of L1CAM Expression
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Mice were anesthetized and perfused with 4% paraformaldehyde; the 25-µm cryostat sections of brain tissue were obtained and stored at −20 °C. Immunohistochemistry for L1CAM was performed by following several optimized steps. Briefly, cell membranes were permeabilized by incubating coverslips in PBS/0.5% Triton X-100 for 5 min. Tissue sections were then blocked in PBS containing 5% bovine serum albumin for 30–60 min at room temperature in a humidified chamber. Sections were then incubated at 4 °C overnight with primary antibody (anti-L1CAM antibody [1:500; ab24345; lot# CR324831-1, Abcam]; anti-Cwh43 antibody [1:200; Sigma; HPA048140; lot# R58111]; anti-acetylated-α tubulin antibody [1:1,000; 5335; lot# 4; Cell Signaling Technology]; rabbit anti-plasmin antibody [OAAB11621; lot# SA100831BD; Aviva Systems Biology]). Sections were then washed in PBS and incubated in the appropriate secondary antibody (anti-mouse IgG [1:2,000; Alexa Fluor 488; A28175; Invitrogen]; goat anti-rabbit IgG [1:2,000; Alexa Fluor 546; A11035; lot #731492; Invitrogen]) for 2 h at room temperature. Tissue sections were again washed three times with PBS, and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) solution (0.2 mg/mL; Sigma-Aldrich). Confocal microscopy was performed using a Leica SP5 II confocal microscope.
3
Evaluating BTZ043 Nanoformulations in THP-1 Cells
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THP-1 cells were cultured as described above and seeded at 1 × 104 cells/well/0.5 mL in 8-well chamber slides (Greiner Bio-One; Kremsmünster, Austria) containing 25 mg/mL PMA. After 72 h, the cells were incubated for 2 h, which dissolved either NP or MP samples at a final BTZ043 concentration of 42 µg/mL in the respective formulation. Treated cells were then washed twice with PBS and fixed with 4% paraformaldehyde (PFA) in PBS for 10 min at RT. For actin staining, cells were incubated for 30 min with 100 µL of 1:1000 Alexa Fluor 488-Phalloidin stock solution (Thermo Fisher Scientific, Waltham, MA, USA) after permeabilization and were blocked for 20 min with 1% w/v BSA and 0.1% w/v saponin in PBS. Nuclei were counterstained with 200 µL of a 1-µg/mL 4′,6-diamidino-2-phenylindole (DAPI) solution (Sigma-Aldrich) for 15 min. Images were taken with a Leica DMi8 Confocal Microscope equipped with a × 63 water immersion objective (HC APO CS2 63 × /1.20), and image analysis was performed with LAS X software (Leica Application Suite X).
4
Immunostaining of Glial and Neuronal Markers
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Cells were fixed by 4% paraformaldehyde for 15 min at room temperature, washed, and blocked in 0.2% Triton X-100 and 10% normal goat serum for 1 h. Cells were then incubated with primary polyclonal rabbit anti-S100 protein antibody (1:100; Abcam, Cambridge, UK), polyclonal mouse anti-p75 antibody (1:50; Abcam), polyclonal chicken anti-GFAP antibody (1:1,000; Abcam), or monoclonal mouse anti-PSA-NCAM antibody (1:200; Merck Millipore, Billerica, MA, USA) overnight at 4°C. Cells were then incubated with indocarbocyanine-conjugated goat anti-chicken (1:1,000; Abcam), indocarbocyanine-conjugated goat anti-mouse (1:1,000; Abcam), or fluorescein-conjugated goat anti-rabbit (1:1,000; Abcam) secondary antibodies for 2 h at 37°C. Thereafter, the cell nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI) solution (1:500; Sigma-Aldrich) for 5 min at room temperature. The primary anti-p75 and anti-S100 antibodies were used for identifying SCs, anti-GFAP antibodies were used for identifying astrocytes, and anti-PSA-NCAM antibodies were used to detect the expression of PSA-NCAM. The results of immunostaining were examined by a fluorescence microscope (FV-1000; Olympus, Tokyo, Japan).
5
Immunohistochemistry for Apoptosis and Proliferation
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For immunohistochemistry, tissues were embedded in OCT compound (Miles Scientific, Elkhardt, IN) and snap-frozen in liquid nitrogen. Frozen sections 6 μm thick were mounted on silane-coated glass slides, and air-dried for 1 h. Cell apoptosis was confirmed by detection of fragmented DNA, using a DeadEnd Colorimetric TUNEL System (Promega, Madison, WI) in accordance with the manufacturer’s instructions. As markers of cell proliferation, sections were stained with anti-Human Ki-67 eFluor 570 (eBioscience, Frankfurt, Germany, 1:200) and anti-SMa-actin (Sigma-Aldrich Japan K.K., Tokyo, Japan, 1:500). Nuclear counter-staining was performed by incubation with 4',6-diamidino-2-phenylindole (DAPI) solution (Sigma-Aldrich, 1 μg/ml in PBS) for 10 min at room temperature. Double-positive cells were counted and averaged for quantitative analysis.
6
NF-κB Translocation Analysis in RAW 264.7 Cells
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The
effect of UaB on LPS-induced nuclear translocation of NF-κB
was assessed using immunofluorescence microscopy. RAW 264.7 cells
were initially grown on glass coverslips for 24 h at 37 °C in
a humidified atmosphere containing 5% CO2 and subsequently
incubated with 80 ng/mL UaB for 1 h prior to treatment with 100 ng/mL
LPS for 30 min in the same culture conditions. The cells were fixed
in 3.7% paraformaldehyde for 15 min, permeabilized with 0.2% triton
X-100 in phosphate-buffered saline (PBS) for 15 min, and blocked for
10 min at room temperature with PBS containing 5% bovine serum albumin
(Sigma-Aldrich, Merck KGaA). The cells were then stained with the
primary antibody from Santa Cruz Biotechnology, Inc. (sc-8008, Dallas,
TX, USA) against NF-κB p65 (1:100 dilution) overnight at 4 °C.
Subsequently, cells were incubated with a fluorescein-conjugated anti-rat
immunoglobulin G (1100 dilution; cat. no. 31629; Molecular Probes,
Thermo Fisher Scientific, Inc.) in the dark for 40 min at 37 °C.
Nuclei were sequentially stained with 2.5 μg/mL 4′,6-diamidino-2-phenylindole
(DAPI) solution (Sigma-Aldrich, Merck KGaA). The slides were then
mounted, and fluorescence images were captured using a fluorescence
microscope (Carl Zeiss, Oberkochen, Germany).
effect of UaB on LPS-induced nuclear translocation of NF-κB
was assessed using immunofluorescence microscopy. RAW 264.7 cells
were initially grown on glass coverslips for 24 h at 37 °C in
a humidified atmosphere containing 5% CO2 and subsequently
incubated with 80 ng/mL UaB for 1 h prior to treatment with 100 ng/mL
LPS for 30 min in the same culture conditions. The cells were fixed
in 3.7% paraformaldehyde for 15 min, permeabilized with 0.2% triton
X-100 in phosphate-buffered saline (PBS) for 15 min, and blocked for
10 min at room temperature with PBS containing 5% bovine serum albumin
(Sigma-Aldrich, Merck KGaA). The cells were then stained with the
primary antibody from Santa Cruz Biotechnology, Inc. (sc-8008, Dallas,
TX, USA) against NF-κB p65 (1:100 dilution) overnight at 4 °C.
Subsequently, cells were incubated with a fluorescein-conjugated anti-rat
immunoglobulin G (1100 dilution; cat. no. 31629; Molecular Probes,
Thermo Fisher Scientific, Inc.) in the dark for 40 min at 37 °C.
Nuclei were sequentially stained with 2.5 μg/mL 4′,6-diamidino-2-phenylindole
(DAPI) solution (Sigma-Aldrich, Merck KGaA). The slides were then
mounted, and fluorescence images were captured using a fluorescence
microscope (Carl Zeiss, Oberkochen, Germany).
7
Nuclear Morphology Visualization by DAPI Staining
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To observe the nuclear morphological changes, the collected cells were fixed with 3.7% paraformaldehyde (Sigma-Aldrich Chemical Co.) in PBS for 10 min at 25°C and air dried. After washing with PBS, the cells were stained with 1 mg/ml of 4',6-diamidino-2-phenylindole (DAPI) solution (Sigma-Aldrich Chemical Co.) at room temperature for 10 min in the dark. Finally, the cells were washed twice with PBS, and the morphological changes in the nucleus were examined using a fluorescence microscope (Carl Zeiss) at ×400 magnification.
8
Immunofluorescence Assay for CD4+ T Cell Subsets
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For immunofluorescence assays, gingival slides were dewaxed and hydrated, and microwave antigen retrieval was performed in ethylenediaminetetraacetic acid buffer (pH 9.0). Following serum blocking (with 4% goat serum for 40 min), the sections were incubated with the following combinations of primary antibodies: anti‐CD4 (GB13064‐1; Servicebio, Wuhan, China) and anti‐IFN‐γ (ab9657; Abcam, Cambridge, UK), anti‐CD4 and anti‐IL‐4 (ab211212; Abcam), anti‐CD4 and anti‐IL‐10 (ab34843; Abcam), and anti‐CD4 and anti‐IL‐17 (ab79056; Abcam) in a 1:100 dilution overnight at 4°C. After primary antibody incubation, the slides were incubated with the following combination of secondary antibodies: Alexa Fluor 488‐conjugated goat anti‐mouse (ab150117; Abcam) and Alexa Fluor 647‐conjugated goat anti‐rabbit (ab150083; Abcam) for 50 min in the dark. Additionally, a control was performed by omitting the primary antibody. Then, the slides were stained with a 1:200 dilution of 4′‐6‐diamidino‐2‐phenylindole (DAPI) solution (Sigma‐Aldrich, Louis, MO, USA) for 10 min in the dark. Finally, the stained cells were visualized by fluorescence microscopy, and images were collected (NIKON ECLIPSE C1, Nikon DS‐U3, Japan).
9
Detecting Apoptotic Cell Morphology
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Cells were stained with 4'-6-diamidino-2-phenylindole (DAPI) solution (Sigma-Aldrich) to confirm the presence of nuclear morphological changes associated with apoptotic cells. Briefly, cells were fixed in 100% ethanol overnight at −20°C, deposited on slides, and stained with DAPI fluorescent dye (2 µg/ml). A fluorescence microscope was used to observe the morphological characteristics of apoptotic cells: nuclear condensation and fragmentation.
10
Isolation and Characterization of Sciatic Nerve Schwann Cells
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SC primary cultures of sciatic nerves and brachial plexus were harvested from postnatal day 1-2 (P1-2) newborn Sprague-Dawley (SD) rats (provided by the Experimental Animal Center of the Fourth Military Medical University) following our established protocols 29 (link). All experimental procedures were conducted under a protocol in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No 85-23, revised1985) and approved by the Animal Research Committee of The Fourth Military Medical University, People's Republic of China. The primary SC cultures were stained by double immunofluorescence using NGF receptor p75 (p75NTR, ab52987; Abcam Inc., UK) and S100 protein (ab52642; Abcam) antibodies. The cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) solution (Sigma-Aldrich). The purity of primary SC cultures was determined by counting the number of p75NTR and S100 double-positive cells and DAPI-labeled cells (Figure S1 ). The final preparations consisted of highly purified (>96%) SCs. The primary SC cultures were passaged no more than 3 times.
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