The expression of CASP4 and CASP9 was detected using IHC in 85 glioma tissue samples. First, we constructed tissue microarrays (TMAs). After dewaxing in xylene, rehydration in alcohol, and the blocking of endogenous peroxidase activity, TMAs were subjected to incubation overnight at 4 °C with specific antibodies against CASP4 (Abcam, AB25898, Waltham, MA, USA) or CASP9 (Abcam, AB202068, Waltham, MA, USA). The next day, a sheep anti-rabbit secondary antibody was supplemented, followed by incubation for half an hour at 25 °C. After DAB was added, color development was observed under a microscope. Finally, hematoxylin was added to counter-stain sections, followed by rinsing with water and dehydration with gradient alcohol as well as fixation using neutral gum sealing tablets. Positive cells in ×400 and ×100 microscopic fields were observed, and two pathologists unaware of the corresponding tissue information were responsible for assessing the IHC results.
Ab25898
Manufactured by Abcam
Sourced in United Kingdom
Ab25898 is a laboratory reagent designed for use in various scientific applications. It serves as a core component in experimental procedures, providing essential functionality to support research activities. The product details and intended uses are presented in a concise, factual, and unbiased manner.
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Lab products found in correlation
Ab40887, by Abcam (3 mentions)
Sc 374615, by Santa Cruz Biotechnology (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Ab202068, by Abcam (1 mentions)
Ab201340, by Abcam (1 mentions)
Ab1872, by Abcam (1 mentions)
A21428, by Thermo Fisher Scientific (1 mentions)
Sc 398715, by Santa Cruz Biotechnology (1 mentions)
Goat anti mouse igg 488, by Thermo Fisher Scientific (1 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
A21422, by Thermo Fisher Scientific (1 mentions)
Ab62721, by Abcam (1 mentions)
Ab203119, by Abcam (1 mentions)
Ab37073, by Abcam (1 mentions)
Ab37152, by Abcam (1 mentions)
Ab207612, by Abcam (1 mentions)
Ab13847, by Abcam (1 mentions)
Ab184909, by Abcam (1 mentions)
Ab21685, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
7 protocols using ab25898
1
Immunohistochemical Analysis of Caspase-4 and Caspase-9 in Glioma
2
Caspase Expression in Dental Tissues
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The slice of EAP that exhibited a clear root canal apex and clinical specimens were selected for staining. HE was performed according to the standard procedure (Biosharp Life Science, Hefei, China).
The immunoreaction sequences were stained by anti-caspase-1, anti-caspase-4, anti-caspase-5, anti-caspase-11 and anti-CD68. The nuclei were visualized by using Hoechst 33342 (Hoechst; ImmunoChemistry Technologies. LLC, Davis, CA, USA). Images were taken by a fluorescence microscope (Leica DM2000, Leica Corporation, Weztlar, Germany). The relative intensity and colocalization of two proteins [Pearson’s correlation coefficient (PCC), overlap coefficient (OC) and scatterplot] were assessed by ImageJ software.
Primary antibody: mouse anti-caspase-1 (sc-398715, Santa Cruz Biotechnology Inc. Dallas, CA, USA), mouse anti-caspase-5 (sc-393346, Santa Cruz Biotechnology); mouse anti-caspase-11 (sc-374615, Santa Cruz Biotechnology); mouse anti-CD68 (ab201340, Abcam, Cambridge, UK); rabbit anti-caspase-1 (ab1872, Abcam); rabbit anti-caspase-4 (ab25898, Abcam); rabbit anti-caspase-5 (ab40887, Abcam).
Secondary antibody: 488 goat anti-mouse IgG (A11011, Invitrogen, Carlsbad, CA, USA), 488 goat anti-rabbit IgG (A11008, Invitrogen), 555 goat anti-mouse IgG (A21422, Invitrogen) and 555 goat anti-rabbit IgG (A21428, Invitrogen).
The immunoreaction sequences were stained by anti-caspase-1, anti-caspase-4, anti-caspase-5, anti-caspase-11 and anti-CD68. The nuclei were visualized by using Hoechst 33342 (Hoechst; ImmunoChemistry Technologies. LLC, Davis, CA, USA). Images were taken by a fluorescence microscope (Leica DM2000, Leica Corporation, Weztlar, Germany). The relative intensity and colocalization of two proteins [Pearson’s correlation coefficient (PCC), overlap coefficient (OC) and scatterplot] were assessed by ImageJ software.
Primary antibody: mouse anti-caspase-1 (sc-398715, Santa Cruz Biotechnology Inc. Dallas, CA, USA), mouse anti-caspase-5 (sc-393346, Santa Cruz Biotechnology); mouse anti-caspase-11 (sc-374615, Santa Cruz Biotechnology); mouse anti-CD68 (ab201340, Abcam, Cambridge, UK); rabbit anti-caspase-1 (ab1872, Abcam); rabbit anti-caspase-4 (ab25898, Abcam); rabbit anti-caspase-5 (ab40887, Abcam).
Secondary antibody: 488 goat anti-mouse IgG (A11011, Invitrogen, Carlsbad, CA, USA), 488 goat anti-rabbit IgG (A11008, Invitrogen), 555 goat anti-mouse IgG (A21422, Invitrogen) and 555 goat anti-rabbit IgG (A21428, Invitrogen).
3
Protein Expression Analysis in Cells
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RIPA buffer lysed the cells to obtain the total protein and a BCA kit was used to quantify the total protein concentration in each group of cells. We separated 30 μg proteins with SDS-PAGE and transferred them to PVDF membranes with semi-dry transfer method. We sealed the PVDF membrane with 50 g/L skim milk powder for 2 h at room temperature. The PVDF membrane was incubated with primary antibody at 4°C overnight, and with secondary antibody at 37°C for 1 h. GAPDH was used as an internal control for the detection of XBP-1 (ab37152, Abcam), LC3I/II (ab62721, Abcam), GRP78 (ab21685, Abcam), ATF4 (ab184909, Abcam), ATF6 (ab203119, Abcam), IRE1 (ab37073, Abcam), CHOP (#2895, Cell Signaling Technology), XBP-1s (#27901, Cell Signaling Technology), XBP-1u (25997-1-AP, ProteinTech), Casp-4 (ab25898, Abcam), casp-3 (ab13847, Abcam), c-casp-3 (ab2302, Abcam), Bax (ab32503, Abcam), Bcl-2 (ab32124, Abcam), Beclin-1 (ab207612, Abcam) and Atg5 (#2630, Cell Signaling Technology). Then, ImageJ software was used for grayscale scanning and quantification.
4
Apoptosis detection using TUNEL assay
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The procedure was performed according to the instructions of the DeadEnd™ Fluorometric TUNEL System (Promega Co., Ltd., Madison, WI, USA). All nuclei were visualized by using Hoechst 33,342. Images were taken by a fluorescence microscope. The TUNEL-positive rate was calculated, PCC, OC and scatterplot (the relative and colocalization of TUNEL and caspase-4/-5/-11 (anti-caspase-4: ab25898, Abcam; anti-caspase-5: ab40887, Abcam; and anti-caspase-11: sc-374615, Santa Cruz Biotechnology)) were assessed by ImageJ [32 (link),33 (link)].
5
Inflammasome Pathway Antibody Validation
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Anti-IL-1β antibody (ab106015), anti-caspase-4 antibody (ab25898), anti-caspase-5 antibody (ab40887), anti-GSDMD antibody -C-terminal (ab228824), and anti-Urotensin II antibody-C-terminal (ab194676) were purchased from Abcam (UK, London, Abcam) used for western blot, IF and IHC methods. Anti-caspase-1 antibody (D7F10) was obtained from Cell Signaling Technology (US, Trask Lane Danvers, CST) and was used in western blot, IF, and IHC. Anti-NLRP -3 antibody (Beijing, China, Wanleibio) was used in IHC and IF, and anti-NLRP-3 antibody (ab91413) was obtained from Abcam for western blot.
6
Colocalization analysis of caspase-4 and APAF-1
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Cells were seeded into a 96-well plate at a density of 1x 10 5 /well and treated as indicated in For colocalization analysis of caspase-4 and APAF-1, cells were washed twice with PBS/T after treatment with 200 μM bile acids for 4 hrs or 1mM LND for 6 hrs, fixed with ice-cold 4% paraformaldehyde for 20 min, permeabilized using 0.2% Triton X-100 in 3% BSA for 15 min, blocked with 3% BSA for 1 hr at room temperature. Then cells were incubated with primary antibodies of caspase-4 (1:50, ab25898, Abcam) and Apaf-1 (1:50, sc-65891, Santa Cruz) at 3% BSA overnight at 4°C.
Washed cells six times with PBS/T (PBS containing Tween-20, 0.05% v/v) before incubating with Donkey Anti-Rabbit IgG(H+L) Antibody, Alexa Fluor 488 (1:1000, A-21206, Invitrogen)
and Donkey Anti-Mouse IgG (H+L) Antibody, Alexa Fluor 555 (1:1000, A-31570, Invitrogen)
for 1 hr at 37 °C in 3% BSA, then washed cells five times with PBS/T. Cell images were acquired on LSM700 confocal microscope (Zeiss, Oberkochen, Germany).
Washed cells six times with PBS/T (PBS containing Tween-20, 0.05% v/v) before incubating with Donkey Anti-Rabbit IgG(H+L) Antibody, Alexa Fluor 488 (1:1000, A-21206, Invitrogen)
and Donkey Anti-Mouse IgG (H+L) Antibody, Alexa Fluor 555 (1:1000, A-31570, Invitrogen)
for 1 hr at 37 °C in 3% BSA, then washed cells five times with PBS/T. Cell images were acquired on LSM700 confocal microscope (Zeiss, Oberkochen, Germany).
7
Immunohistochemical Analysis of CASP4 and CASP8
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