3-μm sections of paraffin-embedded kidneys underwent antigen retrieval by microwave heating in a solution of EnVision FLEX Target Retrieval Solution high-pH (Dako, K800421) for 20 min. Sections were incubated overnight at 4 °C with the following primary antibodies: CD68 (1:200, Abcam ab955), αSMA (1:400, Dako clone 1A4 M0851), FOLR2 (1:200, Invitrogen MA5-26933), TREM2 (1:100, R&D MAB17291) or collagen I (1:400, Invitrogen MA1-26771). Antigen detection was performed with alexa-fluor coupled antibodies: alexa fluor 488 goat anti mouse (for αSMA, Collagen I, CD68, FOLR2, Invitrogen A11001), alexa fluor-555 goat anti rabbit (for FAP, SFRP1, SFRP4 Invitrogen A21428), alexa fluor-647 goat anti rabbit (for FAP, SFRP4 Invitrogen A21245) or alexa fluor-555 goat anti rat (for TREM2 Invitrogen A21434). Nuclei were counterstained with Hoescht before mounting with anti-fade diamond mounting medium (Invitrogen P36970). Images were acquired by an upright widefield Apotome (Zeiss) or LSM 700 Zeiss confocal.
P36970
Manufactured by Thermo Fisher Scientific
Sourced in United States
The P36970 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is a general-purpose device designed for use in scientific research and laboratory settings. The core function of this product is to perform a specific task or operation, but without further details on its intended use or capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Apotome, by Zeiss (2 mentions)
Envision flex target retrieval solution high ph, by Agilent Technologies (1 mentions)
Ab955, by Agilent Technologies (1 mentions)
Lsm 700 confocal, by Zeiss (1 mentions)
Eclipse te2000 confocal microscope, by Nikon (1 mentions)
Cryostat, by Leica (1 mentions)
Goat serum, by Biotium (1 mentions)
Prolong diamond, by Thermo Fisher Scientific (1 mentions)
A27039, by Thermo Fisher Scientific (1 mentions)
Goat serum, by Merck Group (1 mentions)
A1 confocal microscope, by Nikon (1 mentions)
4 protocols using p36970
1
Immunofluorescence Staining of Mouse Kidney
2
Quantification of Viral Gene Expression
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Approximately 48 hours following CED of ZsGreen BPNs, mice were euthanized and transcardially perfused with 10 ml of 2% heparinized 0.9% saline, followed by 10 ml of tris-buffered saline with calcium chloride (0.1 g/liter). Brains were removed, rapidly frozen to −80°C, and cut into 100-μm sections using a cryostat (1905, Leica, Buffalo Grove, IL). Every other section within 2 to 3 mm of the injection site was collected on a slide and mounted with permanent mounting medium (P36970, Invitrogen, Carlsbad, USA). Sections were imaged using a Nikon Eclipse TE2000 confocal microscope (Nikon, Melville, NY) under ×4 magnification. Multiple images were taken and stitched together in montages to capture the entire injection site. Volume of transfected tumor tissue was quantified from these images using a MATLAB script similar to previous studies (32 (link)). Briefly, background fluorescence was subtracted and images were thresholded at 5% of the maximum intensity. The total volume of transgene expression was calculated by multiplying the area of distribution from each slice by the slice thickness and summing values for each slice.
3
Immunofluorescence Staining of Mouse Brain and Primary HBMVECs
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OCT-embedded mouse brain sections were washed in PBS twice and then blocked and permeabilized in 5% donkey serum in PBS with 0.3% Triton X-100 for 1 hour at room temperature. After blocking and permeabilization, slides were incubated with primary antibodies, as described in Supplemental Table 1 , overnight at 4°C, and then washed with PBS 3 times and incubated with proper secondary antibodies at room temperature for 2 hours. After washing in PBS 3 more times, slides were stained with DAPI (0.5 μg/mL) for 10 minutes and mounted with an anti-fade mounting medium (P36970, Invitrogen), and then photographed. Primary HBMVECs (ACBRI 376, Cell Systems) were fixed with 4% PFA for 15 minutes, washed with PBS, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes, and then blocked with blocking buffer for 30 minutes. Primary and secondary antibody application, DAPI staining, mounting, and photographing methods were the same as for tissue staining.
4
Quantification of p62 Positive Fibers in TA
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TA muscles were mounted in OCT, snap frozen, and sectioned in a cryotome at 10 µm thickness. Tissue sections were re-hydrated with three washes of 1X PBS of 5 minutes each. Sections were treated with 0.2% Triton X-100 in 1X PBS for 5 minutes followed by three washes of 1X PBS. Sections were blocked with 0.1% goat serum (Millipore Sigma#G0923) for 1 hour at room temperature followed by overnight primary antibodies incubation (SQSTM1/p62 Rabbit mAb #23214) at 1:200 dilution in 0.1 % goat serum in 4°C. Primary antibodies were washed 3 times with 1X PBS for 5 minutes each followed by incubation with goat anti-rabbit Alexa Fluor 555 (Invitrogen# A27039) 1:300 dilution in 0.1 % goat serum), WGA 488 (100 µg/ml, Biotium #29022), and DAPI at 2 µg/ml for 1 hour at room temperature. Slides were then mounted with Prolong Diamond and coverslipped (Invitrogen #P36970). Whole section images were taken using a Nikon A1 confocal microscope. FIJI was used to quantify the total number of p62positive fibers and minimum Feret diameter.
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