R buffer a

Manufactured by Electron Microscopy Sciences

R-Buffer A is a laboratory reagent used in electron microscopy applications. It serves as a buffer solution, maintaining the pH and ionic conditions required for various sample preparation and analysis procedures. The core function of R-Buffer A is to provide a stable and controlled environment for the samples being examined under an electron microscope.

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Lab products found in correlation

Rm2255 rotary microtome, by Leica camera (1 mentions) Bouin s fixative, by Merck Group (1 mentions) Anti cdh1, by BD (1 mentions) A1 confocal microscope, by Zeiss (1 mentions) 3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions) Hematoxylin gill no 3, by Merck Group (1 mentions) Anti luciferase antibody, by Abcam (1 mentions) Bloxall blocking solution, by Vector Laboratories (1 mentions) Ix73 inverted microscope, by Olympus (1 mentions) Bromodeoxyuridine (brdu), by Santa Cruz Biotechnology (1 mentions) Hep par 1, by Agilent Technologies (1 mentions) Proliferating cell nuclear antigen (pcna), by Cell Signaling Technology (1 mentions) C2 confocal microscope system, by Nikon (1 mentions) Fluorescein system, by PerkinElmer (1 mentions) Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions) Ab71916, by Abcam (1 mentions) Dmi6000 microscope, by Leica camera (1 mentions) Pk 4001, by Vector Laboratories (1 mentions) Sk 4105, by Vector Laboratories (1 mentions) Immpact dab, by Vector Laboratories (1 mentions)

7 protocols using r buffer a

Quantification and Histological Analysis of Lung Metastases

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For quantification of metastatic lesions, mouse lungs were isolated, washed briefly in PBS, fixed in Bouin’s fixative (Sigma) overnight at 4 °C, and stored in 70% ethanol before counting lung nodules. For histological analysis, lungs were fixed in 10% formalin overnight at 4 °C, dehydrated, and embedded in paraffin (Tissue Tek Embedding Station). 3μm sections were cut on a Leica RM2255 rotary microtome. For antigen retrieval, deparaffinized slides were immersed in R-buffer A (Electron Microscopy Services) and boiled in a microwave for 20 min. Samples were blocked and permeabilized for 30 min in blocking buffer (5% normal goat serum, 0.3% TritonX-100 in PBS), followed by primary antibody incubation overnight at 4 °C using anti-CDH1 (1:100, BD Biosciences, 610181). Secondary antibody (1:500, BD Biosciences) was incubated for 1 h at RT. All sections were analyzed using a Nikon A1 confocal microscope and Zeiss fluorescence microscope.

Celià-Terrassa T., Bastian C., Liu D.D., Ell B., Aiello N.M., Wei Y., Zamalloa J., Blanco A.M., Hang X., Kunisky D., Li W., Williams E.D., Rabitz H, & Kang Y. (2018). Hysteresis control of epithelial-mesenchymal transition dynamics conveys a distinct program with enhanced metastatic ability. Nature Communications, 9, 5005.

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Immunohistochemistry of Luciferase in FFPE Tissues

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IHC staining on formalin-fixed, paraffin-embedded tissue sections was performed as before (18 (link)). Briefly, slides were deparaffinized and rehydrated, epitope retrieval was performed in a Retriever 2100 (Aptum Biologics) with R-Buffer A (Electron Microscopy Sciences), and endogenous peroxidases/phosphatases were quenched with BLOXALL blocking solution (Vector Laboratories). Tissues were blocked with Animal-Free Blocker R.T.U. (Vector Laboratories), probed with anti-Luciferase antibodies (Abcam; AB185923, 1:200 dilution) overnight at 4°C, washed with PBS, and incubated with ImmPRESS anti-Rabbit polymer detection reagent (Vector Laboratories) for 30 minutes at room temperature. Visualization was performed by incubation with 3,3′-diaminobenzidine (DAB; Vector Laboratories), tissues were counterstained with Gill No.3 Hematoxylin (Sigma), coverslipped, and imaged on an Olympus IX73 inverted microscope.

Trotter T.N., Wilson A., McBane J., Dagotto C.E., Yang X.Y., Wei J.P., Lei G., Thrash H., Snyder J.C., Lyerly H.K, & Hartman Z.C. (2024). Overcoming Xenoantigen Immunity to Enable Cellular Tracking and Gene Regulation with Immune-competent “NoGlow” Mice. Cancer Research Communications, 4(4), 1050-1062.

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Immunohistochemical Characterization of Liver Tissues

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Livers or tumors were harvested and fixed in 10% (v/v) formalin overnight and embedded in paraffin. Antigen retrieval was performed in R-BUFFER A (Electron Microscopy Sciences) using a vegetable streamer. The following antibodies were used: AE-1 (1:200, Signet Laboratories), Hep Par1 (1:100, Dako), AFP (1:200, Abcam), GFP (1:200, Thermos Fisher Scientific), PCNA (1:10000, Cell Signaling Technology), and BrdU (1:200, Santa Cruz Biotechnology).

Zhao M., Quan Y., Zeng J., Lyu X., Wang H., Lei J.H., Feng Y., Xu J., Chen Q., Sun H., Xu X., Lu L, & Deng C.X. (2021). Cullin3 deficiency shapes tumor microenvironment and promotes cholangiocarcinoma in liver-specific Smad4/Pten mutant mice. International Journal of Biological Sciences, 17(15), 4176-4191.

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Immunohistochemistry Protocol for Phospho-Protein Analysis

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Samples were fixed in 10% formalin and embedded in paraffin. After deparaffinization, slides were washed with 9.83% NaCl for 3 min followed by a PBS wash and a wash in distilled water for 5 min. Heat induced antigen retrieval was performed with pressure cooker (2100 Retriever, PickCell Laboratories B.V.) and R-Buffer A (pH6.0, Electron Microscopy Sciences). Sections were incubated with 2% H₂O₂ in Methanol for 15 minutes for endogenous peroxidase quenching and washed and blocked for non-specific binding in 1% BSA in PBS-Triton 0.3% v/v (PBST) for 1 hour. Subsequently, sections were sequentially incubated with primary antibody at 1:100 dilution for 1 hour, secondary peroxidase goat anti-rabbit IgG antibody (Vector) at 1:250 dilution for 1 hour and Tyramide Signal Amplification (TSA) Fluorescein System (Perkin Elmer, Cat.: NEL701A) according to kit manual. Slides were mounted with Vectashield mounting medium with DAPI (Vector), photographed with Nikon C2 Confocal Microscope system and subsequently stained with Hematoxylin and Eosin. p-AKT (Thr308) p-ERK (Thr202/Tyr204), p-S6 (Ser235/236), and CC3 (Asp 175) were obtained from Cell Signaling, Inc. Ki67 staining was performed as previously described (19 (link)). Quantification of Ki67 staining was performed by scoring the nuclei of cells from each lesion type found in a minimum of ten high-powered fields.

Alagesan B., Contino G., Guimaraes A.R., Corcoran R.B., Deshpande V., Wojtkiewicz G.R., Hezel A.F., Wong K.K., Loda M., Weissleder R., Benes C.H., Engelman J, & Bardeesy N. (2014). Combined MEK and PI3K inhibition in a mouse model of pancreatic cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 21(2), 396-404.

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Immunostaining of Paraffin-Embedded Lung Sections

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Paraffin-embedded lung sections were produced from mice intravenously injected with tumor spheroid single-cell suspensions as described above. Immunostaining was performed targeting Vimentin (Abcam, ab92547, 1:1,000, RRID: AB_10562134), Hmga2 (Cell Signaling Technology, 8179S, 1:500, RRID: AB_11178942), and Epcam (Abcam, ab71916, 1:150, RRID: AB_1603782) using Citrate-based antigen retrieval (Electron Microscopy Sciences, R-Buffer A, 62706-10). All targets were stained using ABC Reagent with biotinylated anti-rabbit secondary antibody (Vector Laboratories, PK-4001) and Immpact DAB (Vector Laboratories, SK-4105) according to product manual. Images were taken using a Leica DMI6000 microscope using a 20X and 5X objective.

Freeburg N.F., Peterson N., Ruiz D.A., Gladstein A.C, & Feldser D.M. (2023). Metastatic Competency and Tumor Spheroid Formation Are Independent Cell States Governed by RB in Lung Adenocarcinoma. Cancer Research Communications, 3(10), 1992-2002.

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Immunohistochemical Staining of Paraffin Sections

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Serial 5‐μm‐thick sections of paraffin‐embedded tissues were deparaffinized, rehydrated, and subjected to antigen retrieval in R‐buffer A (62606‐10; Electron Microscopy Sciences, Hatfield, PA) for 1 hour. After quenching with 3% hydrogen peroxide for 15 minutes, sections were treated with avidin/biotin blocking solution (SP‐2001; Vector Laboratories, Burlingame, CA) for 15 minutes, 3% normal donkey or horse serum and 2.5% Triton X‐100 in phosphate‐buffered saline for 1 hour, and then primary antibodies overnight at 4°C. After washing steps, sections were incubated with appropriate secondary antibodies diluted in phosphate‐buffered saline with 3% serum and 2.5% Triton X‐100 at 37°C for 30 minutes. Sections were treated with peroxidase–avidin complexes (PK‐6106; Vector Laboratories) for 30 minutes at 37°C followed by the substrate 3,3'‐diaminobenzidine tetrahydrochloride. Hematoxylin was used for counterstaining. Antibodies and antigen‐retrieval conditions are summarized in Supporting Table S1.

Lee S., Zhou P., Gupta A, & Shin S. (2018). Reactive Ductules Are Associated With Angiogenesis and Tumor Cell Proliferation in Pediatric Liver Cancer. Hepatology Communications, 2(10), 1199-1212.

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Antigen Retrieval Techniques for Immunohistochemistry

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Antigen retrieval for Aldh1 immunohistochemistry was accomplished using Retrievit 6 (BS-1006-00, BioGenex). For Cytokeratin 19 immunolabeling, antigen retrieval was done by digesting sections with 250 μg/mL proteinase K in 2.5 mmol/L CaCl₂ and 10mmol/L Tris-HCl (pH 7.5) for 6 minutes at room temperature. For NF-kB immunohistochemistry, antigen retrieval was accomplished using R-Buffer A (62706-10, Electron Microscopy Sciences). Antigen retrieval for CD45 was accomplished using a citrate based antigen retrieval buffer and a heat-mediated microwave method. Immunohistochemistry staining was visualized on an Olympus BX40 light microscope.

Hendley A.M., Provost E., Bailey J.M., Wang Y.J., Cleveland M.H., Blake D., Bittman R.W., Roeser J.C., Maitra A., Reynolds A.B, & Leach S.D. (2014). p120 catenin is required for normal tubulogenesis but not epithelial integrity in developing mouse pancreas. Developmental biology, 399(1), 41-53.

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