Advanced oviductal tumors were evaluated for the presence of several immune cell types using standard methods as described previously (26 (link)). Antigen retrieval was performed using Tris-EDTA buffer (pH 9.0) for CD3, CD4 and CD8, and citrate buffer pH 6.0 (Biogenex, San Ramon, CA, USA) for CD45. The primary antibodies used were: rabbit anti-CD3, anti-CD4, anti-CD8 (Abcam, Cambridge, UK), anti-FoxP3 (Cell Signaling, Danvers, MA) and rat anti-Macrophage (Abcam) and anti-CD45 (eBioscience, San Diego, CA, USA).
Citrate buffer ph 6
Manufactured by BioGenex
Sourced in United States
Citrate buffer pH 6.0 is a chemical solution used to maintain a specific pH level in laboratory procedures. It has a pH of 6.0 and is commonly used in various applications such as immunohistochemistry and molecular biology techniques.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880, by Zeiss (2 mentions)
Anti cd45, by Thermo Fisher Scientific (1 mentions)
Anti cd8, by Abcam (1 mentions)
Anti cd4, by Abcam (1 mentions)
Rabbit anti cd3, by Abcam (1 mentions)
Anti foxp3, by Cell Signaling Technology (1 mentions)
3 amino 9 ethylcarbazole, by BioGenex (1 mentions)
I6000, by BioGenex (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by BioGenex (1 mentions)
Alexa fluor 647 donkey anti mouse, by Thermo Fisher Scientific (1 mentions)
Prolong diamond antifade, by Thermo Fisher Scientific (1 mentions)
Pin1 total antibody, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Aqua poly mount, by Polysciences (1 mentions)
Amylo glo, by Biosensis (1 mentions)
Axio scan z1, by Zeiss (1 mentions)
4 protocols using citrate buffer ph 6
1
Immune Cell Profiling in Oviductal Tumors
2
Immunohistochemistry for Antigen Detection
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Immunohistochemistry was performed as previously described [25 (link)]. Briefly, five-micrometer sections were deparaffinized in xylene and rehydrated in graded ethanol. For antigen retrieval, sections were microwaved in citrate buffer pH 6.0 (BioGenex, San Ramon, CA, USA) for 12 min at 95°C and cooled for 30 min prior to immunostaining. Sections were incubated with 3% hydrogen peroxide for 15 min, followed by incubation with primary antibody for 60 min. An automated immunostainer (i6000; BioGenex) was utilized for subsequent incubation steps: sections were incubated in MultiLink biotinylated anti-IgG for 20 min, horseradish peroxidase conjugated secondary antibody for 20 min, followed by development with 3-amino-9-ethyl-carbazole for 10 min (BioGenex). Sections were then counterstained with hematoxylin. All incubation steps were performed at room temperature, and sections were washed with Tris-buffered saline between incubations.
3
Immunofluorescence Microscopy of Paraffin Sections
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Immunofluorescence was done on 5-μm paraffin embedded sections deparaffinized by using a Tissue-Tek slide Stainer in the deparaffinization mode and then antigen retrieved using citrate buffer pH 6.0 (BioGenex, San Ramon, CA) in a microwave oven. Slides were washed 3 × in dH20 and then transferred to 1X TBS. Slides were blocked in 1X TBS, 0.3% triton, and 5% donkey serum. Primary and secondary antibodies were diluted in 1X TBS, 1%BSA, and 0.3% triton. DAPI (Invitrogen) was used to counterstain nuclei. Slides were mounted using Prolong Antifade Diamond (Invitrogen) or Aqua Polymount (Polysciences). Confocal Z-stack images were collected on a Zeiss LSM880 and 0.5 μm optical slices were obtained. Confocal acquisition settings were consistent for image quantification. Pin1 total antibody (mouse, Santa Cruz, sc-46660, 1:100), Pin1 pSer111 (rabbit, Malter Lab, 1:1000), Alexa Fluor 647 donkey anti-mouse (Invitrogen, A31571, 1:1000), Alexa Fluor 568 donkey anti-rabbit (Invitrogen, A10042, 1:1000).
4
Amylo-Glo, GFAP, and Iba1 Immunostaining
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5-μm paraffin sections were cut and mounted on Superfrost Plus slides by the UT Southwestern Molecular Pathology Core. Deparaffinization was done using a Tissue-Tek Slide Stainer in the deparaffinization mode and then antigen was retrieved using citrate buffer pH 6.0 (BioGenex, San Ramon, CA) in a microwave oven. Slides were washed 3 × in dH20 and then transferred to 1X TBS. Sections were stained for Amylo-Glo (Biosensis) per the manufacturer’s directions. Briefly, sections were placed in 1X Amylo-Glo solution for 10 min, then rinsed in 0.9% saline for 5 min, and rinsed in distilled water for 15 s. Slides were then transferred into blocking buffer for immunohistochemistry. Blocking buffer was 5% goat serum, 5% donkey serum, and 0.2% Triton X-100. Primary antibodies used were anti-GFAP (1:1000, Biosensis, C-1373–50) and anti-Iba1 (1:250, Proteintech, 10,904–1-AP). Alexafluor secondary antibodies (A11039, chicken anti-goat 488 and A10042, donkey anti-Rabbit) were used at 1:1000. To-Pro-3 at 1uM (Thermo, T3605) was used to label nuclei per manufacturer’s protocol directions. Whole brain images were collected on a Zeiss Axioscan Z1.
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