Immunofluorescence and immunohistochemistry were performed using a standard protocol 3 (link), 79 (link). In brief, a pressure cooker (Bio SB, #7008) was used for antigen retrieval using antigen unmasking solution (Vector Laboratories, #H-3301) at 95 °C for 30 min. After blocking with 10% goat serum for 1 h, slides were incubated with primary antibodies (1:200 dilution) overnight at 4°C. Slides were then washed with PBS and incubated with secondary antibodies (Invitrogen and CST, 1:500) for 1 hr at room temperature. Slides were then counterstained with DAPI/anti-fade mounting medium (Vector Laboratories, #H-1200-10) for immunofluorescence staining or developed with DAB Quanto (Epredia, #TA125QHDX) followed by hematoxylin for immunohistochemistry staining. Primary antibodies against following proteins were used: STAT3 (CST, #9139S), YAP1 (CST, #14074S), Ki67 (Thermo Fisher, #RM-9106-S0), cleaved caspase 3 (CST, #9661S), F4/80 antibody (CST, #70076S), LDHA (CST, #2012), and Mac-2 (Biolegend, #125403).
Dapi anti fade mounting medium
Manufactured by Vector Laboratories
DAPI/anti-fade mounting medium is a solution used for preserving and protecting fluorescent-labeled samples mounted on microscope slides. It contains the nuclear stain DAPI (4',6-diamidino-2-phenylindole) and an anti-fade agent to enhance and stabilize fluorescent signals.
Automatically generated - may contain errors
Lab products found in correlation
Antigen unmasking solution, by Vector Laboratories (3 mentions)
Dab quanto, by Epredia (3 mentions)
Pressure cooker, by Bio SB (2 mentions)
Did fluorescent dye, by Biotium (2 mentions)
Ax ax r confocal microscope system, by Nikon (1 mentions)
Nbp1 30042, by Novus Biologicals (1 mentions)
Confocal microscope, by Nikon (1 mentions)
Horse serum, by American Type Culture Collection (1 mentions)
Axio scope a1 microscope, by Zeiss (1 mentions)
Alexa fluor 488 goat anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
Lab tek culture slides, by Thermo Fisher Scientific (1 mentions)
J61899, by Thermo Fisher Scientific (1 mentions)
P7949, by Merck Group (1 mentions)
H 1500, by Vector Laboratories (1 mentions)
Axiocam mrm camera, by Zeiss (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
8 ohdg, by Santa Cruz Biotechnology (1 mentions)
Dmi8 microscope, by Leica (1 mentions)
Antigen unmarking solution, by Vector Laboratories (1 mentions)
Dfc345 fx, by Leica (1 mentions)
9 protocols using dapi anti fade mounting medium
1
Immunostaining protocol for protein analysis
2
Immunodetection Techniques for Tissue Analysis
3
Tracking EV Uptake in Tumor Cells
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EVs were labeled with DiD fluorescent dye (Biotium, #60014) for 30 min on a shaker. Then, the DiD-labeled EVs were added to tumor cell culture medium and incubated for 24 hrs at 37 °C. Cells were fixed with 4% PFA for 15 min and counterstained with 4ʹ,6-diamidino-2-phenylindole (DAPI)/anti-fade mounting medium (Vector Laboratories, #H-1200-10) before confocal microscope (Nikon) examination.
4
Immunofluorescence and Immunohistochemistry Protocol
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Immunofluorescence and immunohistochemistry were performed using a standard protocol3 (link),79 (link). In brief, a pressure cooker (Bio SB, #7008) was used for antigen retrieval using antigen unmasking solution (Vector Laboratories, #H-3301) at 95 °C for 30 min. After blocking with 10% goat serum for 1 h, slides were incubated with primary antibodies (1:200 dilution) overnight at 4 °C. Slides were then washed with PBS and incubated with secondary antibodies (Invitrogen and CST, 1:500) for 1 hr at room temperature. Slides were then counterstained with DAPI/anti-fade mounting medium (Vector Laboratories, #H-1200-10) for immunofluorescence staining or developed with DAB Quanto (Epredia, #TA125QHDX) followed by hematoxylin for immunohistochemistry staining. Primary antibodies against following proteins were used: STAT3 (CST, #9139 S), YAP1 (CST, #14074 S), Ki67 (Thermo Fisher, #RM-9106-S0), cleaved caspase 3 (CST, #9661 S), F4/80 antibody (CST, #70076 S), LDHA (CST, #2012), and Mac-2 (Biolegend, #125403).
5
Fluorescent Labeling of Extracellular Vesicles
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EVs were labeled with DiD fluorescent dye (Biotium, #60014) for 30 min on a shaker. Then, the DiD-labeled EVs were added to tumor cell culture medium and incubated for 24 hrs at 37 °C. Cells were fixed with 4% PFA for 15 min and counterstained with 4,6-diamidino-2-phenylindole (DAPI)/anti-fade mounting medium (Vector Laboratories, #H-1200-10) before confocal microscope (Nikon) examination.
6
Immunofluorescence Imaging of Transfected Cells
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HEK-293 T transfected cells were cultured on 8 wells Lab-Tek culture slides (Nunc, C7182). 15 h or 48 h after transfection, cells were washed once with 200 µL of Phosphate-Buffered Saline 1× (prepared from PBS 10× pH 7.4 Gibco, 70,011–036) and fixed in 4% paraformaldehyde solution (Alfa Aesar, J61899) for 15 min. Cells were further washed three times with 200 µL of PBS 1× and permeabilized with 200 µL of PBS 1× supplemented with 0.2% Tween 20 (Sigma, P7949) for 15 min. After 30 min of incubation in 200 µL of blocking solution made with PBS 1×, 0.2% Tween 20 and 2.5% horse serum (ATCC, 30–2040), cells were incubated with 50 µg/mL of primary antibodies GN_mAb_Env01 diluted in blocking solution for 1 h. After 3 washes with 200 µL of PBS 1×, cells were incubated for 1 h with 4 µg/mL Alexa Fluor 488 goat anti-mouse IgG antibody (ThermoFisher, A11029) diluted in blocking solution. After 3 washes with 200 µL of PBS 1×, cells were counterstained with DAPI/anti-fade mounting medium (Vectashield, H-1500). Microscopy was performed with a Zeiss AXIO Scope A.1 microscope, equipped with Zeiss AxioCam MRm camera. Composite images were created using ImageJ software.
7
Quantifying DNA Damage Markers
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Cells were seeded at 1.5 × 10 5 per well in a six-well plate and treated with 100 nM IPI-9119 or DMSO. On the fourth day, cells were reseeded at 7500 cells per chamber in eight-chamber glass slides coated with poly-l-lysine. Cells were incubated for an additional 48 hours with IPI-9119 or vehicle before fixing in formaldehyde for 15 min at room temperature. Slides were washed with phosphate-buffered saline (PBS), and cell permeabilization was performed (0.5% Triton X-100), followed by blocking with BSA. Phosphorylated H2A.X (Ser 139 ) (CST, 80312), 53BP1 (CST, 4937), and 8-OHdG (Santa Cruz, sc-393871) antibodies were incubated overnight at 4°C, followed by 1-hour incubation with goat anti-rabbit immunoglobulin G (IgG) Alexa Fluor 594 (ab150080) and goat anti-mouse Alexa Fluor 488 (ab150113). Slides were mounted in DAPI Antifade Mounting Medium (Vectashield). Imaging was carried out on a THUNDER imager LEICA DMi8 Microscope (LEICA, Germany), using the software LASx (3.7.4.23463). Image quantification data were performed using ImageJ software (National Institutes of Health).
8
Multicolor Immunofluorescence Staining of FFPE Samples
9
Cryopreservation and Immunohistochemistry of Spinal Cord Tissue
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Tissues were cryopreserved by incubation in 20% sucrose until tissue sank (1–2 days),
frozen in the TissueTek cutting medium (Sakura Finetek, Torrance, CA). The spinal cords
were cut into longitudinal sections 30 µm in thickness with cryostat (Leica CM1900).
Unspecific binding sites were blocked by incubation with (PBS, 5% FBS, 0.5% Triton
X-100) for 2 hours at room temperature. Primary and secondary antibodies were suspended
in (PBS, 1% FBS, 0.1% Triton X-100). Sections were incubated overnight at 4° C with
primary antibodies: profilin1 (1:1000, Sigma), GFAP (1:1000; Novus Biologicals
NB-300-141), and IBA1 (1:1000; Wako 019-19741) and 2 hours at room temperature with
secondary antibodies (i.e., 1:500, Alexa-Flour 488 or 647; Invitrogen). F/G-actin ratio
was assayed by staining tissues with phalloidin (1:200, Sigma) to detect F-actin and
DNaseI conjugates (1:200, ThermoFisher) to detect G-actin. Sections were mounted onto
glass slides with DAPI/anti-fade mounting medium (Vector Laboratories). Images were
taken with Zeiss Confocal Microscope Confocal LSM 510 (Zeiss, Thornwood, NY).
Quantification of fluorescence intensity was analysed by ImageJ.
frozen in the TissueTek cutting medium (Sakura Finetek, Torrance, CA). The spinal cords
were cut into longitudinal sections 30 µm in thickness with cryostat (Leica CM1900).
Unspecific binding sites were blocked by incubation with (PBS, 5% FBS, 0.5% Triton
X-100) for 2 hours at room temperature. Primary and secondary antibodies were suspended
in (PBS, 1% FBS, 0.1% Triton X-100). Sections were incubated overnight at 4° C with
primary antibodies: profilin1 (1:1000, Sigma), GFAP (1:1000; Novus Biologicals
NB-300-141), and IBA1 (1:1000; Wako 019-19741) and 2 hours at room temperature with
secondary antibodies (i.e., 1:500, Alexa-Flour 488 or 647; Invitrogen). F/G-actin ratio
was assayed by staining tissues with phalloidin (1:200, Sigma) to detect F-actin and
DNaseI conjugates (1:200, ThermoFisher) to detect G-actin. Sections were mounted onto
glass slides with DAPI/anti-fade mounting medium (Vector Laboratories). Images were
taken with Zeiss Confocal Microscope Confocal LSM 510 (Zeiss, Thornwood, NY).
Quantification of fluorescence intensity was analysed by ImageJ.
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