Total genomic gDNA was isolated from log-phase promastigotes of each parasite cell line using the Wizard Genomic DNA purification kit (Promega Corporation, Madison, WI, USA) according to manufacturer’s recommendations. For Southern blot analysis, such gDNA was digested with several restriction endonucleases and separated on 1 % agarose gels, transferred to positively charged Nylon membranes (Roche), and cross-linked to the membranes by UV irradiation using a Stratalinker® 2400 (Stratagene, Stratagene La Jolla, CA, USA). Subsequently, blots were hybridized under high stringency using a DIG-labeled DNA probe (i.e., LdINV-DIG856), corresponding to a portion of the L. donovani invertase ORF (i.e., nt 85–942). After high stringency washing (0.1× SSC, 0.1 % SDS at 65 °C), the hybridized fragments were visualized using an anti-digoxigenin-alkaline phosphatase conjugated antibody in conjunction with a chemiluminescent reagent (CSPD) according to manufacturer’s instructions (Roche). Images were captured from such blots using BIOMAX™-MR X-ray film (Kodak, Rochester, NY, USA) and processed using Adobe Photoshop CS2 (Adobe). In addition, DNA was also isolated from the LdINV-16.3 cosmid clone and similarly digested with several different restriction endonucleases. Such preparations were subsequently subjected to Southern blot analysis as above.
Biomax mr x ray film
Manufactured by Kodak
Sourced in United States
Biomax MR X-ray films are a line of high-quality X-ray films designed for medical and scientific applications. These films are used in various laboratory settings to capture and record images from X-ray exposures. The films provide reliable and consistent results, making them a reliable choice for various imaging needs.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal chemiluminescence kit, by Thermo Fisher Scientific (3 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (2 mentions)
Nylon membrane, by Roche (1 mentions)
Wizard genomic dna purification kit, by Promega (1 mentions)
Horseradish peroxidase labeled anti mouse antibodies, by Merck Group (1 mentions)
Hybond c nitrocellulose membrane, by Cytiva (1 mentions)
Total akt, by Cell Signaling Technology (1 mentions)
Phospho akt ser473, by Cell Signaling Technology (1 mentions)
Denhardt s solution, by Merck Group (1 mentions)
Polyadenylic acid, by Merck Group (1 mentions)
Bio dot microfiltration apparatus, by Bio-Rad (1 mentions)
Her2 polyclonal antibody, by Agilent Technologies (1 mentions)
Hnpp fast red fluorescent detection set, by Roche (1 mentions)
Nbt bcip, by Roche (1 mentions)
Imagequant version 5, by GE Healthcare (1 mentions)
Gs 800 scanner, by Bio-Rad (1 mentions)
11 protocols using biomax mr x ray film
1
Genomic DNA Isolation and Southern Blot Analysis
2
Western Blot Analysis of Protein Expression
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Equal protein amounts (50 μg) of each sample were resolved by 4-12% NuPAGE® Novex Bis-Tris polyacrylamide gel, and electroblotted to nitrocellulose membrane using iBlot™ Dry Blotting System (Invitrogen, Carlsbad, CA). The membranes were blocked with 5% nonfat dry milk in TBST for 60 min, washed, and subsequently incubated overnight at 4 °C in TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween-20) with 5% BSA containing the following antibodies; anti-MUC 1 (diluted 1:1000; Mouse monoclonal, Novocastra), anti-EMA (diluted 1:1000; Mouse monoclonal, DAKO) or anti-GAPDH (diluted 1:5000; Mouse monoclonal, Calbiochem). Specific bindings were detected with horseradish peroxidase-labeled anti-mouse antibodies (Chemicon International, Temecula, CA) and enhanced with SuperSignal Chemiluminescence kit (Pierce Biotechnology). Signals were detected on KODAK BIOMAX MR X-ray film (Kodak, Rochester, NY).
3
Akt Signaling Pathway and sPLA2-IIA Inflammation
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To verify the role of Akt, the specific inhibitor LY294002 (Alexis, Lausen, Switzerland) was added into the culturing medium for 6 h co-incubation. Western blot analysis was conducted to evaluate phosphorylated and total Akt in order to investigate if this pathway is involved in the proinflammatory effect of sPLA2-IIA. Aliquot of protein extracted from the cells (40 μg) was mixed with 0.25 volume of loading buffer (250 mmol/l Tris-HCl, pH 6.8, 10% (w/v) SDS, 25% (w/v) glycerol, 0.2% (w/v) bromophenol blue and 5% (v/v) β-mercaptoethanol), boiled for 5 min at 95°C and then subjected to 10% SDS-polyacrylamide gels. After electrophoresis, the proteins were transferred to Hybond-C nitrocellulose membrane (Amersham Pharmacia Biotech UK Ltd, Bucks, UK). The membrane was incubated with polyclonal antibodies against phospho-Akt (Ser473, 1:1000) or total-Akt (1:1000) (Cell Signaling Technology, Beverly, MA, USA) at 4°C with gentle agitation overnight. Peroxidase-conjugated AffiniPure Donkey Anti-rabbit IgG (H+L) secondary antibody (1:5000, Promega, Madison, WI, USA) was used after washing. The immunostains were visualized by ECL Western Blotting Detection Reagent (Amershanm BioSciences, Piscataway, NJ, USA), and then exposed to Hyperfilm. The relative intensities of the bands were detected on Biomax MR X-ray film (Kodak, Nanjing, China).
4
In-Situ Hybridization for Rat Brain
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Hybridization was performed as described by Stocker and Pedarzani (2000 (link)). In brief, slides were air dried and hybridized overnight at 42°C in 100 μl solution containing 50% formamide, 10% dextran sulfate, 50 mM dithiothreitol (DTT), 300 mM NaCl, 30 mM Tris-HCl (pH 7.4), 4 mM EDTA, 1× Denhardt's solution (Sigma), 0.5 mg/ml acid/alkali denatured salmon sperm DNA, and 0.5 mg/ml polyadenylic acid (Sigma). After hybridization, slides were washed twice for 20 minutes each in 1× SSC containing 50 mM β-mercaptoethanol, for 45 minutes in 1× SSC at 57°C, and for 5 minutes in 1× SSC, followed by 5 minutes in 0.1× SSC at room temperature. Sections were once again dehydrated in an ascending ethanol series, air dried, and exposed to Kodak Biomax MR X-ray film (Eastman Kodak, Rochester, NY) for 14 days. For cellular resolution, slides were subsequently dipped in photographic emulsion Kodak NTB, incubated for 12–20 weeks at 4°C, and then developed in Kodak D-19 for 3.5 minutes. Sections were counterstained with 0.1% cresyl violet (Nissl stain) to confirm cytoarchitecture and analyzed via bright-and darkfield microcopy. Rat brain regions were identified according to Paxinos et al. (1994 ), Altman and Bayer (1995 ), and Paxinos and Watson (1998 ).
5
Western Blot Quantification of FeSOD
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The blots were destained in 100% methanol, rinsed
in Tween-20 tris
buffer saline (TTBS), and blocked for 2 h in 5% nonfat Dry Milk (NFDM)
in TTBS. The blots were then incubated overnight in primary antibody
(anti-FeSOD [Agrisera, Cat# AS06125] diluted 1:20,000 in 2% NFDM TTBS)
and rinsed for 3 × 10 min in TTBS. The blots were placed in the
secondary antibody (anti-Rabbit IgG-HRP [SeraCare, Cat# 5220–0337]
diluted 1:20,000 in 2% NFDM TTBS) for 2 h, rinsed as above, treated
with enhanced chemiluminescence, and exposed to Kodak BioMax MR X-ray
film. Western blot films (KO5401 #1–3: Kodak BioMax MR 3-min
exposures) were scanned with a laser densitometer (Model PDSI, Molecular
Dynamics Inc, Sunnyvale, CA). The scanner was checked for linearity
prior to scanning with a calibrated Neutral Density Filter Set (Melles
Griot, Irvine, CA). Band volume above background (manual marking)
was quantified using Phoretix 1D software (version 11.2) with a Windows
10 compatible computer.
in Tween-20 tris
buffer saline (TTBS), and blocked for 2 h in 5% nonfat Dry Milk (NFDM)
in TTBS. The blots were then incubated overnight in primary antibody
(anti-FeSOD [Agrisera, Cat# AS06125] diluted 1:20,000 in 2% NFDM TTBS)
and rinsed for 3 × 10 min in TTBS. The blots were placed in the
secondary antibody (anti-Rabbit IgG-HRP [SeraCare, Cat# 5220–0337]
diluted 1:20,000 in 2% NFDM TTBS) for 2 h, rinsed as above, treated
with enhanced chemiluminescence, and exposed to Kodak BioMax MR X-ray
film. Western blot films (KO5401 #1–3: Kodak BioMax MR 3-min
exposures) were scanned with a laser densitometer (Model PDSI, Molecular
Dynamics Inc, Sunnyvale, CA). The scanner was checked for linearity
prior to scanning with a calibrated Neutral Density Filter Set (Melles
Griot, Irvine, CA). Band volume above background (manual marking)
was quantified using Phoretix 1D software (version 11.2) with a Windows
10 compatible computer.
6
Quantitative Western Blot Analysis of FFPE Tissue
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The protein lysates (10 μg) extracted from human breast FFPE tissue sections was dotted to 0.2 μm nitrocellulose membrane using Bio-Dot Microfiltration Apparatus (Bio-Rad, Hercules, CA). The membranes were blocked in 5% nonfat dry milk in TTBS (50 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.5) for 1 hour at room temperature (RT), washed, and then incubated overnight in 5% BSA in TTBS at 4°C containing antibodies; HER-2 polyclonal antibody (DAKO, 1:2000) and anti-GAPDH (Calbiochem, Gibbstown, NJ, 1:1000). Subsequently, the membranes were washed and incubated with horseradish peroxidase labeled anti-rabbit and anti-mouse secondary antibody for 90 minutes and enhanced using a SuperSignal Chemiluminescence kit (Pierce Biotechnology) and detected on Kodak Biomax MR X-ray film (Kodak).
7
In Situ Hybridization of Brain Tissue
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In situ hybridization was performed as previously described [11] (link). Briefly, animals were sacrificed by cervical dislocation and fresh brain tissue was dissected out, embedded in Tissue Tek (Sakura), frozen on dry ice and stored at −80°C until used. 14 µm cryostat sections were cut and hybridization was performed according to protocols using either digoxigenin-, fluorescein- or 35S-labelled antisense RNA probes. In digoxigenin-labelled insitu hybridization experiments, NBT/BCIP (Roche) was used as substrate for alkaline phosphatase, while in radioactive insitu hybridization sections were exposed to Biomax MR X-ray films (Kodak) for two to seven days. For double ISH, sections were hybridized simultaneously with DIG- and fluorescein-labelled probes. A two-step chromogenic reaction using NBT/BCIP and HNPP/Fast Red Fluorescent Detection Set (Roche) was performed to visualize DIG- and fluorescein-labelled riboprobes. Specimens were counterstained with DAPI.
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In situ hybridization was performed as previously described [11] (link). Briefly, animals were sacrificed by cervical dislocation and fresh brain tissue was dissected out, embedded in Tissue Tek (Sakura), frozen on dry ice and stored at −80°C until used. 14 µm cryostat sections were cut and hybridization was performed according to protocols using either digoxigenin-, fluorescein- or 35S-labelled antisense RNA probes. In digoxigenin-labelled insitu hybridization experiments, NBT/BCIP (Roche) was used as substrate for alkaline phosphatase, while in radioactive insitu hybridization sections were exposed to Biomax MR X-ray films (Kodak) for two to seven days. For double ISH, sections were hybridized simultaneously with DIG- and fluorescein-labelled probes. A two-step chromogenic reaction using NBT/BCIP and HNPP/Fast Red Fluorescent Detection Set (Roche) was performed to visualize DIG- and fluorescein-labelled riboprobes. Specimens were counterstained with DAPI.
8
In Situ Hybridization of Dopamine Receptors in Mice
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Mice were killed and their brains quickly dissected, embedded in Tissue Tek (Sakura) and frozen at 80 °C. Fourteen-micrometre cryostat sections were cut in the coronal plane and in situ hybridization analyses were performed according to protocols previously described.37 (link) Coronal sections along the rostro-caudal extent of striatum and substantia nigra from both B6 and BTBR male adult mice were hybridized with 35S-labelled antisense riboprobes (Drd1, 1.3 Kb: B6 n=4, BTBR n=7; Drd2, 0.4 Kb: B6 n=4, BTBR n=7). Hybridized sections were exposed to Biomax MR X-ray films (Kodak, Rochester, NY, USA) for 2–8 days.
9
Western Blot Analysis of Protein Samples
10
In-Situ Hybridization of RASD2 in Macaque Brains
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ISH analysis was performed on coronal sections from adult macaque brains (n = 3 control group), according to a protocol previously described [12 (link)]. RASD2 cDNA was cloned by PCR using the following primers: forward 5’-TACCAGCTGGACATCCTGG-3’ , reverse 5’-CGTCACCGTACTGCACGG-3’ . RASD2 35S-labelled antisense riboprobe, 433 bp in length (nucleotides 398–830, XM-015150119), was used ISH analysis. Hybridized sections were exposed to Biomax MR X-ray films (Kodak, Rochester, NY) for three days. Images of autoradiography films of radioactive ISH experiments were scanned at a resolution of 4800 dpi and images at high magnification were acquired using bright field light microscopy [34 (link)].
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