Formalin-fixed, paraffin-embedded PCa tissues were cut into 4-μm-thick sections, deparaffinized with xylene, washed with gradient ethanol, and then rehydrated in double-distilled water. Soaking in 3% H2O2 for 15 min inhibited the activity of endogenous peroxidase. The slides were heated in citrate buffer to achieve antigen retrieval. The slides were washed in phosphate-buffered saline (PBS) and blocked with 10% goat serum. After incubation with an anti-TLR9 rabbit polyclonal antibody (1:200, ab37154, Abcam, Cambridge, MA, USA) or an anti-VEGF-C rabbit polyclonal antibody (1:300, bs-1586R, BIOSS Antibodies, Beijing, China) at 4°C overnight, the sections were incubated with a biotin-labeled secondary antibody (1:200, ab205718, Abcam). Finally, the sections were counterstained with hematoxylin. Liver tissue was used as a positive control for TLR9 staining, while colon cancer tissue was used for VEGF-C. PBS was used as a negative control.
Ab37154
Manufactured by Abcam
Sourced in United States
Ab37154 is a recombinant monoclonal antibody targeting Histone H3. This product is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
Ab27478, by Abcam (2 mentions)
Peroxidase conjugated goat anti rabbit igg, by Agilent Technologies (2 mentions)
Ab205718, by Abcam (1 mentions)
Talin 1, by Merck Group (1 mentions)
Myd88, by Santa Cruz Biotechnology (1 mentions)
Lamin b1, by Santa Cruz Biotechnology (1 mentions)
Ab85803, by Abcam (1 mentions)
Alexa 594 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Streptavidin biotin blocking solution, by Vector Laboratories (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Alexa 488 conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Chicken serum, by Merck Group (1 mentions)
P0448, by Agilent Technologies (1 mentions)
3 amino 9 ethylcarbazole aec substrate, by Vector Laboratories (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Aec substrate, by Vector Laboratories (1 mentions)
7 protocols using ab37154
1
Immunohistochemical Analysis of TLR9 and VEGF-C in Prostate Cancer
2
Western Blotting Analysis of Immune Signaling
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Western blotting (immunoblotting) was performed according to standard protocols. The following antibodies were used: TLR3 (6961), TLR4 (14358), MyD88 (4283), TIRAP (13077), TRIF (4596), IRAK4 (4364), TRAF3 (4729), Vav1 (4657), Vav1 (2502), p85 (4257), PIP5K1c (3296), p85 (4292), pERK1/2 T202/Y204 (4370), ERK1/2 (4695), pAKT S473 (4060), pAKT T308 (13038), pJNK1/2 T183/Y185 (9255), pp38 T180/Y182 (9211), pIRF3 S396 (4947), pIκBα S32 (2859), FAK (3285), EEA-1 (3288), pTBK1 S172 (5483), TBK1 (3504; all from CST); CD11b M1/70 (ab184308), CD29 (ab183666), TLR9 (ab37154; all from Abcam); TLR4 (sc-293072), TLR3 (sc-32232), GFP (sc-9996), GFP (sc-8334), Vav (sc-132), MyD88 (sc-74532), MyD88 (sc-136970), Na/K ATPase (sc-21712), AKT (sc-1618), JNK1/2 (sc-7345), p38 (sc-7972), IRF3 (sc-376455), lamin B1 (sc-374015), p65 (sc-109), CD29 (sc-8978), IκBα (sc-1643; all from Santa Cruz Biotechnology); TRAM (AF4348; R&D Biosystems), TRAF6 (GTX113029; GeneTex), Talin1 (T3287), Flag M2 (F3165 and F7425; both from Sigma-Aldrich); GAPDH (AM4300), IRAK4 (700026), and TLR4 (48–2300; all from Invitrogen).
3
Immunohistochemical Analysis of Placental TLR9
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To evaluate TLR9 expression in placental tissues, IHC staining was performed. Placental tissue sections deparaffinized using xylene and a series of ethanol gradients (75%-100%). The sections were then immersed in a 0.01 M citrate solution, followed by thermal antigen repair. Endogenous enzyme activity was blocked using 1%periodate. The sections were incubated with a TLR9 antibody (1:200, Abcam, ab37154) overnight at 4°C, then co-incubated with a secondary antibody (1:100, Abiowell, AWS0003) for 30 min. Lastly, the sections were sealed with neutral gel and transferred under a microscope for observation and image acquisition (magnification 100× and 400×). The Image-Pro-Plus software was used to determine positive rates and the TLR9 expression levels.
4
Immunohistochemical Analysis of TLR9 in HCC
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This study enrolled patients with HCC from the Taipei Medical University Shuang-Ho Hospital, Taipei, Taiwan. The study was reviewed and approved by the hospital’s institutional review board (TMU-JIRB: 201302016). Collected samples were fixed in 4% paraformaldehyde and embedded in paraffin, with 5 μm thick sections cut from the paraffin blocks. Staining was performed using anti-TLR9 (1:5000; cat. no. ab37154, Abcam), followed by secondary antibody and hematoxylin and eosin (H&E).
5
Immunofluorescent Staining of Paraffin-Embedded Tissues
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Paraffin-embedded tissues were deparaffinized and dehydrated using a graded ethanol series. Antigen retrieval was performed by boiling the slides in 10 mM citrate buffer (pH 6.0) for 4 minutes. Endogenous peroxidase activity was blocked by incubation with 3% H2O2 for 10 minutes, followed by washing in PBS. Sections were incubated in streptavidin-biotin blocking solution for 1 hour (Vector Laboratories, Burlingame, CA, USA). Sections were stained with the anti-IFN-α monoclonal antibody (E-7; Santa Cruz), anti-IFN-β monoclonal antibody (ab85803; Abcam) and anti-TLR9 monoclonal antibody (ab37154; Abcam). Alexa 488-conjugated anti-mouse IgG and Alexa 594-conjugated anti-rabbit IgG (Invitrogen) were then applied as secondary antibodies. 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) was used at a concentration of 300 nM for nuclear counterstaining. The resulting stained specimens were mounted in VectaShield Antifade Mounting Medium (CH-1000; Vector Laboratories). The slides were then observed under a fluorescence microscope (Leica).
6
Immunocytochemical Analysis of TLR9 in Adipocytes
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3T3-L1 fibroblasts were differentiated into mature adipocytes as described previously. The shock-frozen sections were fixed with ice cold acetone (ROTH, Karlsruhe, Germany) for immunocytochemistry. Air-dried cells were incubated in PBS for rehydration. Endogenous peroxidase activity was blocked with 3% H 2 O 2 (ROTH). To avoid non-specific protein binding, cells were incubated in 10% bovine serum albumin (BSA, ROTH), 10% fetal calf serum (FCS, Sigma-Aldrich) and 10% chicken serum (Sigma-Aldrich), followed by 3-h incubation in a moist chamber with rabbit anti-mouse TLR9 antibody (5 µg/mL in 1% BSA; ab37154; Abcam). Cells were then stained with peroxidase-conjugated goat anti-rabbit IgG (2.5 µg/mL in 1% BSA; P0448; DAKO) for 90 min. Color development with 3-amino-9-ethylcarbazole (AEC) substrate (Vector Laboratories, Burlingame, CA, USA) at room temperature was stopped after microscopic examination. Rabbit isotype-matched IgG sera (ab27478; Abcam) served as an isotype control. Parallel experiments without primary antibody were carried out as negative controls.
7
Immunohistochemical Detection of TLR9 in Adipose Tissue
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Formalin-fixed murine and human adipose tissue was embedded in paraffin. Paraffin was removed with xylene (ROTH) and rehydrated by consecutive washing steps with 100, 96 and 70% ethanol (ROTH), aqua dest. and PBS. Endogenous peroxidase activity was blocked with 3% H 2 O 2 . Tissue samples were incubated for 60 min in citrate buffer at 60°C. Non-specific binding sites were blocked with 5% BSA for 60 min in a humid chamber followed by an overnight incubation with rabbit anti-mouse/human TLR9 antibody (10 µg/mL in 1% BSA; ab37154; Abcam). Tissues were then stained with peroxidase-conjugated goat anti-rabbit IgG (2.5 µg/mL in 1% BSA; DAKO P0448) for 90 min. Color development with AEC substrate (Vector Laboratories) at room temperature was stopped after microscopic examination. Rabbit isotype-matched IgG sera (ab27478; Abcam) served as an isotype control. Parallel experiments without primary antibody were carried out as negative controls.
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