Tumor biopsies were embedded in OCT medium containing cryomolds and immediately frozen in 2-methyl-butane. 5 μm frozen sections of the tissues were made using the cryostat and layered on superfrost® plus slides (Thermo Scientific, Rockford, IL). The slides were fixed in 4% para-formaldehyde for 15 minutes, washed and blocked for 60 minutes at room temperature. The slides were then stained for 3 hours at room temperature (RT) with antibodies for CXCL10 (ab9807) and CXCL8 (ab18672), both from Abcam, Cambridge, MA. The slides were washed 5 times with 1 × PBS and incubated with secondary antibodies anti-rabbit-Alexa 488, anti-mouse-Alexa 647 (both from Cell Signal, Danvers, MA) and with nuclear dye Sytox orange (Invitrogen, Carlsbad, CA) for 30 minutes at RT. The slides were washed 5 times with 1 × PBS. Cover slips were mounted on the sections using prolong gold anti-fade solution (Life Technologies). Confocal analyses of stained slides were performed using a LEICA TCS SL DMRE Microsystems.
Ab9807
Manufactured by Abcam
Sourced in United Kingdom
Ab9807 is a lab equipment product. It is a monoclonal antibody that can be used for various laboratory applications. The core function of this product is to bind to a specific target molecule, but no further details on its intended use can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold antifade solution, by Thermo Fisher Scientific (1 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (1 mentions)
Sytox orange, by Thermo Fisher Scientific (1 mentions)
Ab18672, by Abcam (1 mentions)
Envision system hrp labeled polymer anti rabbit, by Agilent Technologies (1 mentions)
Ab9797, by Abcam (1 mentions)
Ab9955, by Abcam (1 mentions)
Ab9720, by Abcam (1 mentions)
4 protocols using ab9807
1
Multiplexed Immunofluorescence of Tumor Biopsies
2
Immunohistochemical Analysis of Gingival and DF Tissues
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For immunohistochemical staining, gingival tissue and DF tissue were fixed in 10% buffered formalin for 1 day, embedded in paraffin, and then sectioned at a thickness of 3 μm. The specimens were subjected to IHC staining with antibodies specific for CXCL10 (rabbit polyclonal, diluted 1 : 50; Ab9807, Abcam, Cambridge, UK), CSTA (rabbit polyclonal, diluted 1 : 2,000; Ab61223, Abcam), AMBN (rabbit polyclonal, diluted 1 : 200; Ab116347, Abcam), and CXCL12 (rabbit polyclonal, diluted 1 : 50; Ab9797, Abcam). Endogenous peroxidase activity was quenched via addition of 3% hydrogen peroxide. The sections were incubated in 5% bovine serum albumin to block nonspecific binding. The primary antibodies were diluted to facilitate optimal staining, and the sections were incubated overnight. After incubation, EnVision+ System HRP-Labeled Polymer anti-rabbit (K4003, Dako North America, Inc., CA, USA) was applied for 20 min. Color development was performed using labeled streptavidin biotin kits (Dako) according to the manufacturer's instructions.
3
Immunohistochemical Analysis of Tissue
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IHC analysis was performed as described previously.8 Briefly, FFPE blocks were prepared from 2 to 3 various parts of the tissue piece. Immune cells were assessed using an antibody recognizing CD45 (DAKO, mouse monoclonal antibody (clone 2B11+ PD7/26); dilution: 1:400) and IP10 with an anti-IP10 (Abcam, rabbit polyclonal (ab9807); dilution: 1:100). Standardization of the dilution, incubation, and development times appropriate for each antibody allowed an accurate comparison of expression levels in all cases. Positive and negative control slides were analyzed in parallel, with the latter incubated with PBS instead of primary antibodies.
4
Evaluation of Chemokine Expression in Tumor Samples
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Tissue microarrays were constructed as previously described [28 (link)]. Namely, formalin-fixed, paraffin-embedded tumor specimens were reviewed histologically using hematoxylin and eosin staining and two duplicate1.0-mm tissue cores from different areas were used to construct the TMA. Anti-CXCL9 CXCL10 and CXCL11 antibodies (1:100; ab9720, ab9807 and ab9955; Abcam, Cambridge, MA) were used for IHC staining. The negative controls were performed without primary antibodies. Two pathologists blinded to the clinical data evaluated the staining of each specimen. To avoid the inter-observer variability, the mean value of scores was adapted for further analysis. The staining was evaluated by semi-quantitative immunoreactivity score system, deriving from the multiplication of intensity of immunohistochemical staining (0, no staining; 1, weak; 2, moderate and 3, strong) and percentage of positive cells (1 point for each 10% increment; ranges from 1 to 10) ranges from 0 to 30. More than medium value was considered as high expression.
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