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Quantifying Tumor-Associated Tertiary Lymphoid Structures

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In I.P. tumors grown in C57BL/6, CXCR5^−/−, LTβR-Ig and checkpoint treated mice, and in S.C. tumors containing PDPN⁺ CAF or mouse embryonic fibroblasts, intratumoral TA-TLS were identified as tight aggregates of 50 or more B cells in juxtaposition to PNAd⁺ vasculature and associated with a reticular network of elongated PDPN⁺ CAF that are not tightly apposed to tumor endothelium (Rodriguez et al., 2018 (link)). T cells and DC were either intermingled with B cell aggregates (non-classical structure) or organized as a separate zone (classical structure). Using an AxioImager with Apotome (Zeiss) and ImageJ Software (NIH) or Imaris image analysis software (Bitplane), B cell and PDPN staining pattern were used to draw a boundary of the TA-TLS, and quantitate its surface area.
In I.P. tumors grown in μMT^−/−, TNFR1/2^−/−, or in I.P. tumors grown in Rag1^−/− mice repleted with bulk CD8 T cells and/or B cells, we identified tight clusters of CD45⁺ –hematopoietic cells associated with a network of elongated PDPN⁺ CAF that were not tightly apposed to tumor endothelium as elements of TA-TLS development. Using an AxioImager with Apotome (Zeiss) and ImageJ Software (NIH), clusters of CD45⁺ and PDPN⁺ staining pattern is used to draw a boundary and quantitate its surface area.

Rodriguez A.B., Peske J.D., Woods A.N., Leick K.M., Mauldin I.S., Meneveau M.O., Young S.J., Lindsay R.S., Melssen M.M., Cyranowski S., Parriott G., Conaway M.R., Fu Y.X., Slingluff CL J.r, & Engelhard V.H. (2021). Immune mechanisms orchestrate tertiary lymphoid structures in tumors via cancer-associated fibroblasts. Cell reports, 36(3), 109422.

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Imaging Protocols for Tissue Analysis

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Immunostaining and in situ hybridization on sections were imaged using either a Zeiss AxioImager equipped with the HXP 120V fluorescence light source, a Hamamatsu Orca Flash 4.0 LT B&W camera and Zeiss AxioCam 105 color camera, or with a Leica DM5000B equipped with Leica EL 6000 metal halide light source and Hamamatsu Orca-Flash4.0 V2 sCMOS camera. Whole-mount immunofluorescence staining was imaged using a Zeiss AxioImager with the Zeiss ApoTome application. Whole-mount imaging for intact kidney size measurements was performed using a Leica MZFLIII equipped with a Leica DFC7000T camera.

Li H., Kurtzeborn K., Kupari J., Gui Y., Siefker E., Lu B., Mätlik K., Olfat S., Montaño-Rodríguez A.R., Huh S.H., Costantini F., Andressoo J.O, & Kuure S. (2021). Postnatal prolongation of mammalian nephrogenesis by excess fetal GDNF. Development (Cambridge, England), 148(10), dev197475.

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Immunocytochemistry and Histochemistry Protocols for hPSC, KER, and FIB Cells

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For immunocytochemistry, hPSC, KER, and FIB were fixed in 4% paraformaldehyde (Euromedex) and permeabilized with 0.1% Triton X-100 (Sigma, St. Louis, MI, USA). After blocking non-specific interactions with 5% bovine serum albumin (Sigma-Aldrich), samples were incubated with primary antibodies overnight at +4 °C in blocking buffer. After washing, samples were incubated 1 h with species-specific fluorophore-conjugated secondary antibodies and counterstained with DAPI at 1 μg/mL (Millipore) to allow nuclei detection. Image acquisition was performed with an inverted microscope (Axio Imager, Zeiss, Oberkochen, Germany) or the HighContentScreening module CellInsight CX7 (Thermo Fisher Scientific, Waltham, MA, USA), after cell segmentation and thresholding was based on negative control (secondary antibody only).
For immunohistochemistry and Hematoxylin Eosin staining (HE), tissues were fixed in 10% formaline (VWR) before paraffin embedding. All IHC staining was carried out on paraffin sections of 4 µm thickness using Ventana Discovery XT IHC module according to the manufacturer’s instructions. HE image acquisition was performed with EVOS™ XL Core Imaging System (Thermo Fisher Scientific). IHC image acquisition was performed with an inverted microscope (Axio Imager, Zeiss). The list of antibodies is presented in Table S2.

Domingues S., Darle A., Masson Y., Saidani M., Lagoutte E., Bejanariu A., Coutier J., Ayata R.E., Bouschbacher M., Peschanski M., Lemaitre G, & Baldeschi C. (2022). Clinical Grade Human Pluripotent Stem Cell-Derived Engineered Skin Substitutes Promote Keratinocytes Wound Closure In Vitro. Cells, 11(7), 1151.

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Immunohistochemical Analysis of Rat Brain

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Rat tissues were prepared for immunostaining as previously described [14 (link)]. Sections were incubated with anti-CNTF (1:000; R&D systems), anti-CNTFRα (1:000; R&D systems), anti-FLAG (1:2000; Sigma), or anti-tyrosine hydroxylase (TH, 1:2000; Pel-Freez, Rogers, AR) overnight at 4°C and then incubated with biotin-conjugated secondary antibodies, followed by an avidin-biotin complex kit (Vector Laboratories, Burlingame, CA). The signal was detected by incubating sections in 0.5 mg/ml 3,3’-diaminobenzidine (DAB; Sigma) in 0.1 M phosphate buffer (PB; Sigma) containing 0.003% H₂O₂. Sections were analyzed under a microscope (Axio Imager; Carl Zeiss, Göttingen, Germany). For immunofluorescence labeling, brain sections were incubated overnight with one of the following pairs: anti-FLAG (1:2000; Sigma) and anti-TH (1:2000; Pel-Freez), anti-TH (1:2000; Pel-Freez) and anti-CNTF (1:000; R&D systems), or anti-TH (1:2000; Pel-Freez) and anti-CNTFRα (1:000; R&D systems). The next day, sections were incubated with Cy3- (1:200; Millipore, Bedford, MA) and FITC-conjugated IgG (1:200; Millipore) for 1 h, and then washed and mounted with Vectashield mounting medium (Vector Laboratories). Sections were analyzed under a bright-field microscope (Axio Imager; Carl Zeiss) or with confocal microscopy (LSM700; Carl Zeiss).

Jeong K.H., Nam J.H., Jin B.K, & Kim S.R. (2015). Activation of CNTF/CNTFRα Signaling Pathway by hRheb(S16H) Transduction of Dopaminergic Neurons In Vivo. PLoS ONE, 10(3), e0121803.

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Confocal Microscopy Imaging Protocol

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Except where noted, confocal images were acquired using a CCD digital camera (OrcaR2 Hamamatsu Photonics) and a spinning disk confocal microscope (CSU-10; Yokogawa) mounted on a compound microscope (Axioimager; Carl Zeiss) with a Plan-APOCHROMAT 100x/1.4 oil differential interference contrast objective and controlled by either iVision software (Biovision Technologies) or μManager⁶⁰. All other images were acquired using a CCD digital camera (Axiocam mrm; Carl Zeiss) mounted on a microscope (Axioimager; Carl Zeiss) with a Plan-APOCHROMAT 100x/1.4 oil differential interference contrast objected controlled by either Axiovision 4.6 (Carl Zeiss) or Zen 2012 (Carl Zeiss). Acquired images were processed using ImageJ 1.46r and Photoshop (CS5.1; Adobe). For microscopy, worms were anesthetized on 5% agar pads containing 0.01M NaN3. To avoid bias, for each individual experiment, images were acquired in a random order.

Matus D.Q., Chang E., Makohon-Moore S.C., Hagedorn M.A., Chi Q, & Sherwood D.R. (2014). Cell Division and Targeted Cell Cycle Arrest Opens and Stabilizes Basement Membrane Gaps. Nature communications, 5, 4184.

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Quantifying Tumor-Associated Tertiary Lymphoid Structures

Cited 1 time

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In I.P. tumors grown in C57BL/6, CXCR5^−/−, LTβR-Ig and checkpoint treated mice, and in S.C. tumors containing PDPN⁺ CAF or mouse embryonic fibroblasts, intratumoral TA-TLS were identified as tight aggregates of 50 or more B cells in juxtaposition to PNAd⁺ vasculature and associated with a reticular network of elongated PDPN⁺ CAF that are not tightly apposed to tumor endothelium (Rodriguez et al., 2018 (link)). T cells and DC were either intermingled with B cell aggregates (non-classical structure) or organized as a separate zone (classical structure). Using an AxioImager with Apotome (Zeiss) and ImageJ Software (NIH) or Imaris image analysis software (Bitplane), B cell and PDPN staining pattern were used to draw a boundary of the TA-TLS, and quantitate its surface area.
In I.P. tumors grown in μMT^−/−, TNFR1/2^−/−, or in I.P. tumors grown in Rag1^−/− mice repleted with bulk CD8 T cells and/or B cells, we identified tight clusters of CD45⁺ –hematopoietic cells associated with a network of elongated PDPN⁺ CAF that were not tightly apposed to tumor endothelium as elements of TA-TLS development. Using an AxioImager with Apotome (Zeiss) and ImageJ Software (NIH), clusters of CD45⁺ and PDPN⁺ staining pattern is used to draw a boundary and quantitate its surface area.

Rodriguez A.B., Peske J.D., Woods A.N., Leick K.M., Mauldin I.S., Meneveau M.O., Young S.J., Lindsay R.S., Melssen M.M., Cyranowski S., Parriott G., Conaway M.R., Fu Y.X., Slingluff CL J.r, & Engelhard V.H. (2021). Immune mechanisms orchestrate tertiary lymphoid structures in tumors via cancer-associated fibroblasts. Cell reports, 36(3), 109422.

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7

Quantifying Subcellular GFP Fluorescence

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Z-stacks of ser2prom3>DMA-1::GFP and ser2prom3>HPO-30::GFP fluorescence in distal, middle and tail region of mid-L4 stage worms were taken using a spinning disk confocal microscope (Axio Imager; Carl Zeiss) with a 100x Plan-Aprochromat objective lens (1.4 NA). Number of vesicular units (either single vesicles or vesicle clusters) was quantified from images of maximum intensity projections generated from z-stack images using ImageJ. The size of each image was 76.8μm x 76.8 μm.
Z-stacks of ser2prom3>GFP::RAB-10 fluorescence in middle region of mid-L4 stage worms were taken using a spinning disk confocal microscope (Axio Imager; Carl Zeiss) with a 63x Plan-Aprochromat objective lens (1.4 NA). Number of vesicular units (either single vesicles or vesicle clusters) was quantified from maximum intensity projections generated from z-stacks using ImageJ. The size of each image was 88.1μm x 88.1 μm.

Zou W., Yadav S., DeVault L., Jan Y.N, & Sherwood D.R. (2015). RAB-10-Dependent Membrane Transport Is Required for Dendrite Arborization. PLoS Genetics, 11(9), e1005484.

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Confocal Imaging and Data Processing

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Confocal images were acquired using a microscope (Axio Imager; Carl Zeiss) with a 100× Plan-Apochromat objective lens (1.4 NA) equipped with an EM charge-coupled device (CCD) or ORCA-R2 CCD camera (Hamamatsu Photonics) and a spinning disc confocal scan head (CSU-10; Yokogawa Electric Corporation) controlled by iVision software (Biovision Technologies) or Micro-Manager software (Edelstein et al., 2010 (link)). The point-spread function (PSF) of imaging GFP fluorescence on the confocal microscopes used was calculated and well within the acceptable range. Compound light and fluorescent microscopy images were acquired using a microscope (AxioImager; Carl Zeiss) with a 100× Plan-Apochromat objective lens (1.4 NA) and with a CCD camera (AxioCam MRm; Carl Zeiss) controlled by Axiovision software (Carl Zeiss). Acquired images were processed using ImageJ 1.40 and Photoshop CS6 Extended software (Adobe Systems Inc.). 3D reconstructions were built from confocal z stacks, analyzed, and exported using Imaris 7 (Bitplane). Movies were annotated using Photoshop software (Adobe Systems Inc.). Graphs and figures were built using Illustrator software (CS6 Extended; Adobe Systems Inc.).

Wang Z., Linden L.M., Naegeli K.M., Ziel J.W., Chi Q., Hagedorn E.J., Savage N.S, & Sherwood D.R. (2014). UNC-6 (netrin) stabilizes oscillatory clustering of the UNC-40 (DCC) receptor to orient polarity. The Journal of Cell Biology, 206(5), 619-633.

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Immunostaining of Mouse Brain Tissues

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Mice tissues for immunostaining were prepared as previously described [16 (link), 21 (link)], and the following primary antibodies were used for immunohistochemistry: anti-neuronal nuclei (NeuN, 1 : 200, Millipore, Temecula, CA) and anti-ionized calcium-binding adapter molecule 1 (Iba1, 1 : 2000, Wako Pure Chemical Industries, Japan) overnight at 4°C and then incubated with biotin-conjugated secondary antibodies, followed by an avidin-biotin complex kit (Vector Laboratories, Burlingame, CA). The signal was detected by incubating sections in 0.5 mg/mL 3,3′-diaminobenzidine (DAB, Sigma) in 0.1 M phosphate buffer (PB, Sigma) containing 0.003% H₂O₂. Sections were analyzed under a microscope (Axio Imager; Carl Zeiss, Göttingen, Germany). For immunofluorescence labeling, brain sections were incubated overnight with one of the following pairs: anti-NeuN (1 : 200, Millipore) and anti-LC3B (1 : 1000, Cell Signaling, Beverly, MA); anti-Iba1 (1 : 2000, Wako Pure Chemical Industries) and anti-TNFα (1 : 500, R&D Systems, Minneapolis, MN). The next day, the sections were incubated with Cy3- (1 : 200, Millipore) and FITC-conjugated IgG (1 : 200, Millipore) and then washed and mounted with Vectashield mounting medium (Vector Laboratories). The stained samples were analyzed under a bright-field microscope (Axio Imager) or a confocal microscopy (LSM700, Carl Zeiss, Germany).

Jeong K.H., Jung U.J, & Kim S.R. (2015). Naringin Attenuates Autophagic Stress and Neuroinflammation in Kainic Acid-Treated Hippocampus In Vivo. Evidence-based Complementary and Alternative Medicine : eCAM, 2015, 354326.

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Confocal Microscopy Imaging Protocol

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Except where noted, confocal images were acquired using a CCD digital camera (OrcaR2 Hamamatsu Photonics) and a spinning disk confocal microscope (CSU-10; Yokogawa) mounted on a compound microscope (Axioimager; Carl Zeiss) with a Plan-APOCHROMAT 100x/1.4 oil differential interference contrast objective and controlled by either iVision software (Biovision Technologies) or μManager⁶⁰. All other images were acquired using a CCD digital camera (Axiocam mrm; Carl Zeiss) mounted on a microscope (Axioimager; Carl Zeiss) with a Plan-APOCHROMAT 100x/1.4 oil differential interference contrast objected controlled by either Axiovision 4.6 (Carl Zeiss) or Zen 2012 (Carl Zeiss). Acquired images were processed using ImageJ 1.46r and Photoshop (CS5.1; Adobe). For microscopy, worms were anesthetized on 5% agar pads containing 0.01M NaN3. To avoid bias, for each individual experiment, images were acquired in a random order.

Matus D.Q., Chang E., Makohon-Moore S.C., Hagedorn M.A., Chi Q, & Sherwood D.R. (2014). Cell Division and Targeted Cell Cycle Arrest Opens and Stabilizes Basement Membrane Gaps. Nature communications, 5, 4184.

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Axio imager

Lab products found in correlation

679 protocols using axio imager

Quantifying Tumor-Associated Tertiary Lymphoid Structures

Imaging Protocols for Tissue Analysis

Immunocytochemistry and Histochemistry Protocols for hPSC, KER, and FIB Cells

Immunohistochemical Analysis of Rat Brain

Confocal Microscopy Imaging Protocol

Quantifying Tumor-Associated Tertiary Lymphoid Structures

Quantifying Subcellular GFP Fluorescence

Confocal Imaging and Data Processing

Immunostaining of Mouse Brain Tissues

Confocal Microscopy Imaging Protocol

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