Collagen 4

Manufactured by Bio-Rad

Sourced in Japan

Collagen IV is a type of extracellular matrix protein that is a key structural component of basement membranes. It plays a crucial role in cell adhesion, migration, and differentiation. Collagen IV is often used in cell culture and tissue engineering applications.

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Lab products found in correlation

Lsm 700 confocal microscope, by Zeiss (2 mentions) Alexa fluor 488, by Thermo Fisher Scientific (1 mentions) Lama 1, by Merck Group (1 mentions) Sp8 microscope, by Leica camera (1 mentions) Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions) Lsm700, by Leica camera (1 mentions) Click it edu alexa fluor 647 imaging kit, by Thermo Fisher Scientific (1 mentions) Vectashield, by Vector Laboratories (1 mentions) Carbonic anhydrase 9, by Santa Cruz Biotechnology (1 mentions) Hif 1α, by Novus Biologicals (1 mentions) Cleaved caspase 3, by Cell Signaling Technology (1 mentions) β actin, by Santa Cruz Biotechnology (1 mentions) Isolectin ib4, by Thermo Fisher Scientific (1 mentions) H 1200 10, by Vector Laboratories (1 mentions) Mec13, by BD (1 mentions) Bx60 microscope, by Olympus (1 mentions) C2 upright confocal microscope, by Nikon (1 mentions) Caveolin 1, by Cell Signaling Technology (1 mentions)

7 protocols using collagen 4

Retinal Flatmount Immunostaining

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Mouse eyes were fixed in 4% paraformaldehyde for 30 min and remained in PBS overnight before dissecting. The neuroretina was peeled away from the RPE and incisions made to flatten before fixing in ice-cold methanol. Flatmounts were blocked and permeabilised for 1h at RT in 2X PBS containing 1% bovine serum albumin, 3% Triton X-100, 0.5% Tween-20 and 0.12% sodium azide. Flatmounts were stained using Collagen IV (Rabbit polyclonal, AbD Serotec, 1:500) antibody overnight at RT, followed by anti-rabbit IgG conjugated to AlexaFluor 488 and anti-rat IgG conjugated to AlexaFluor 594 (1:200 dilution). Sections were again washed and incubated with 1μg/ml DAPI for 2 min at RT before mounting in Mowiol mounting medium. The entire ribbon containing the optic nerve was analysed for each sample. We did not detect noticeable differences in C3 or C3 breakdown products along the ribbon, and therefore took images halfway along on either side of the optic nerve.

Williams J.A., Stampoulis D., Gunter C.E., Greenwood J., Adamson P, & Moss S.E. (2016). Regulation of C3 Activation by the Alternative Complement Pathway in the Mouse Retina. PLoS ONE, 11(8), e0161898.

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Histological Characterization of Mouse Tissues

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Following PBS and PFA perfusion, mouse tissues were incubated with 30% sucrose solution overnight, embedded in frozen section media and snap frozen on dry ice. 16 μm sections were cut on a Micron cryostat for histology staining and quantifications. The following antibodies were used for immunostainings: Podocalyxin (AF1556 R&D; 1:250), Collagen IV (2150-1470 Serotec; 1:250), SPP1 (HPA027541, Atlas antibodies 1:250), COL6A1 (HPA019142, Atlas antibodies, 1:250) and LAMA1 (L9393, Sigma, 1:500). Secondary goat antibodies conjugated to Alexa Fluor 488, 555, 594 or 647 were purchased from Life Technologies and used at 1:500. Images were acquired using Zeiss LSM 700 confocal microscope and Zen software.

Månberg A., Skene N., Sanders F., Trusohamn M., Remnestål J., Szczepińska A., Aksoylu I.S., Lönnerberg P., Ebarasi L., Wouters S., Lehmann M., Olofsson J., Antequera I.V., Domaniku A., De Schaepdryver M., De Vocht J., Poesen K., Uhlén M., Anink J., Mijnsbergen C., Vergunst-Bosch H., Hübers A., Kläppe U., Rodriguez-Vieitez E., Gilthorpe J.D., Hedlund E., Harris R.A., Aronica E., Van Damme P., Ludolph A., Veldink J., Ingre C., Nilsson P, & Lewandowski S.A. (2021). Altered perivascular fibroblast activity precedes ALS disease onset. Nature medicine, 27(4), 640-646.

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Quantitative Analysis of Retinal Angiogenesis in Mice

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For quantitative analysis of angiogenesis, eyes were harvested from P7 mice and fixed in 4% PFA on ice for 2 h. After fixation, eyes were washed in PBS and then dissected to remove the retinal tissue. Following dissection, retinas were incubated in blocking buffer (3% normal goat serum, 1% BSA, 0.5% Triton X-100 and 0.5% Tween-20) for 1 h at RT and then incubated with primary antibodies diluted in incubation buffer (1.5% normal goat serum, 0.5% BSA, 0.25% Triton X-100 and 0.25% Tween-20) overnight at 4 °C. Primary antibodies against the following proteins were used: PECAM (1:200, #AF3628, R&D Systems), ERG (1:200, #ab92513, Abcam), Collagen IV (1:400, #2150-1470, AbD Serotec). After primary antibody incubation, retinas were washed four times in 0.1% PBS Triton X-100 and incubated with Alexa Fluor-conjugated secondary antibodies (Thermo, 1:250) for 2 h at RT. Following secondary antibody staining, retinas were washed four times in 0.1% PBS Triton X-100 and the Click-iT EdU Alexa Fluor 647 imaging kit was used for detection of EdU incorporation (Thermo). Retinas were mounted on glass slides using Vectashield (Vector Labs) and imaging was performed using a Zeiss LSM700 or Leica SP8 microscope. All images were acquired without prior knowledge of the genotype of the individual samples.

Stone O.A., El-Brolosy M., Wilhelm K., Liu X., Romão A.M., Grillo E., Lai J.K., Günther S., Jeratsch S., Kuenne C., Lee I.C., Braun T., Santoro M.M., Locasale J.W., Potente M, & Stainier D.Y. (2018). Loss of pyruvate kinase M2 limits growth and triggers innate immune signaling in endothelial cells. Nature Communications, 9, 4077.

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Quantitative Histological Analysis of Tumor Samples

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H&E, picrosirus red, IHC, and IF staining were performed using standard techniques on formalin-fixed paraffin-embedded sections. Tissues for quantitative evaluation were processed in parallel. For quantification, whole tissue sections were imaged on a Keyence BD microscope with an automated stage. The whole virtual slide was used for quantification using the ImageJ software package (rsbweb.nih.gov/ij/).
Quantification of PSR staining was performed using ImageJ. RGB (Red,Green,Blue) images were split in the three-color channels. The green channel was used for quantification of the relative area that displayed a signal above a certain, constant threshold.
Antibodies used for IHC/IF or WB: cleaved caspase-3 (Cell Signaling Technology Cat# 9661, RRID:AB_2341188), Carbonic Anhydrase IX (Santa Cruz Biotechnology Cat# sc-25599, RRID:AB_2066539)), Hif1α (Novus Cat# NB100–131H, RRID:AB_1108863), CD31 (Santa Cruz Biotechnology Cat# sc-28188, RRID:AB_2267979), CD34 (Abcam Cat# ab8158, RRID:AB_306316), Collagen IV (Bio-Rad / AbD Serotec Cat# 2150-1470, RRID:AB_2082660), Ki-67 (Abcam Cat# ab16667 RRID:AB_302459), LOX (IMGENEX Cat# IMG-6442A RRID:AB_1930256), LOXL2 (Biorbyt Cat# orb41134 RRID:AB_10987961), β-Actin (Santa Cruz Biotechnology Cat# sc-1615 RRID:AB_630835).

Rossow L., Veitl S., Vorlová S., Wax J.K., Kuhn A.E., Maltzahn V., Upcin B., Karl F., Hoffmann H., Gätzner S., Kallius M., Nandigama R., Scheld D., Irmak S., Herterich S., Zernecke A., Ergün S, & Henke E. (2018). LOX-catalyzed collagen stabilization is a proximal cause for intrinsic resistance to chemotherapy. Oncogene, 37(36), 4921-4940.

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Immunostaining of Mouse Eye Tissues

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Mouse eyes were collected and fixed in 4% paraformaldehyde for 1 hour. Dissected choroid or frozen section was permeabilized in PBS containing 5% normal rabbit serum albumin, 2% bovine serum albumin, and 0·3% Triton X-100 for 1 hour. The following antibodies were used: isolectin IB4 (ThermoFisher Scientific, I21411, RRID: AB_23146), IBA1 (Wako, 019-19741, RRID: AB_839504), SOCS3 (Cell Signalling, 2923, RRID: AB_2255132), Collagen IV (Bio-Rad, 2150-1470, RRID: AB_2082644), vWF (Thermo Fisher, PA5-16634, RRID: AB_10982615), GFP (Abcam, ab13970, RRID: AB_300798), CD31 (MEC 13·3, BD Biosciences, 550274, RRID: AB_393571), and VEGF receptor 2 (VEGFR2) (clone Avas12, BioLegend, 136406, RRID: AB_2044067). DAPI in an anti-fade mounting medium (Vector Labs, H-1200-10) was used for nuclear staining.

Wang T., Zhou P., Xie X., Tomita Y., Cho S., Tsirukis D., Lam E., Luo H.R, & Sun Y. (2021). Myeloid lineage contributes to pathological choroidal neovascularization formation via SOCS3. EBioMedicine, 73, 103632.

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Immunofluorescence Staining of Murine Eye Tissues

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Mice were euthanized, and eyes collected and fixed in 2% paraformaldehyde for 1 hr before transferring into cold PBS. For staining of endothelial or pericyte markers eyes were fixed in ice cold methanol. After dissection of the various compartments of the eye (choroid, retina, iris) the tissues were incubated in 20 mM EDTA at 37°C for 30 minutes. Tissues were then incubated in a solution containing 0.3% Triton-X, 2% bovine serum albumin and 10% of normal goat serum in PBS at room temperature for 30 min. Tissues were incubated with the primary antibody overnight at 4°C followed by incubation with the secondary antibody at room temperature for 1 hr. Detection of the immediate-early (IE1) protein of MCMV was performed with the 6/58/1 monoclonal antibody [53 (link)]. Monoclonal antibodies specific for the following cell surface markers were used to stain tissue sections: MHC-class II (clone M5/114), CD45 (clone 30F11), CD31 (clone Mec 13.3), PDGFRβ (clone APB5), F4/80 (clone BM8), CD4 (clone RM4-5), CD8 (clone 53–6.7), IBA1 (Wako Pure Chemicals Industry, Osaka, Japan), CollagenIV (Biorad-2150-1470). Slides were counterstained with Hoechst to visualize nuclei. Assessment of stained specimens was performed using an epifluorescence microscope (Olympus BX60 microscope: Olympus, Tokyo, Japan) or a Nikon C2 Upright Confocal microscope.

Voigt V., Andoniou C.E., Schuster I.S., Oszmiana A., Ong M.L., Fleming P., Forrester J.V, & Degli-Esposti M.A. (2018). Cytomegalovirus establishes a latent reservoir and triggers long-lasting inflammation in the eye. PLoS Pathogens, 14(5), e1007040.

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Immunohistochemical Analysis of Brain Vasculature

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For string vessel measurements, pepsin antigen retrieval was performed for 5 min at 37 °C (0.1 mg ml⁻¹ pepsin in PBS, 0.2 N HCl) on 50 μm thick brain sections. For vascular density, an antigen retrieval procedure (20 min at 95 °C in 10 mM sodium citrate solution) was performed before the staining. Sections were blocked with 3% BSA in PBS containing 0.1% Triton X-100 for 6 h at room temperature, and incubation with primary antibodies (collagen IV: Bio-Rad, #134001, 1:200; caveolin-1: Cell Signaling Technology, #3267, 1:400) was performed at 4 °C overnight, while incubation with secondary antibodies was performed in blocking solution at room temperature for 2 h. Images were taken using a confocal laser scanning microscope (Leica, SP5). For all analyses, we imaged four fields from at least two sections per mouse and analysis was performed blinded using ImageJ.

Cecon E., Fernandois D., Renault N., Coelho C.F., Wenzel J., Bedart C., Izabelle C., Gallet S., Le Poder S., Klonjkowski B., Schwaninger M., Prevot V., Dam J, & Jockers R. (2022). Melatonin drugs inhibit SARS-CoV-2 entry into the brain and virus-induced damage of cerebral small vessels. Cellular and Molecular Life Sciences, 79(7), 361.

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