Key Product Details

Validated by

Knockout/Knockdown

Species Reactivity

Validated:

Human, Mouse, Rat, Fish, Primate

Cited:

Human, Mouse, Rat, Fish

Predicted:

Bovine (100%). Backed by our 100% Guarantee.

Applications

Validated:

Immunohistochemistry, Immunohistochemistry-Paraffin, Western Blot, Flow Cytometry, Flow (Intracellular), Immunocytochemistry/ Immunofluorescence, Simple Western, Immunoprecipitation

Cited:

Immunohistochemistry-Paraffin, Western Blot, Immunocytochemistry/ Immunofluorescence, Simple Western, Immunoprecipitation, IF/IHC

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Format

BSA Free
View all DRP1 Primary Antibodies »

Product Specifications

Immunogen

A synthetic peptide made to an internal region within residues 500-600 of the human DRP1 protein. [Swiss-Prot# O00429]

Reactivity Notes

Use in Rat reported in scientific literature (PMID:34622072). Fish reactivity reported in scientific literature (PMID: 25008790).

Localization

Cytoplasm; cytosol. Intracytoplasmic membrane; peripheral mambrane protein. Mainly cytosolic. Also membrane-associated. Localizes to mitochondia at spots of division events. Associated with peroxisomal membranes, it is recruited in part by PEX11B.

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Theoretical MW

81 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Scientific Data Images for DRP1 Antibody - BSA Free

Western Blot: DRP1 AntibodyBSA Free [NB110-55288]

Western Blot: DRP1 AntibodyBSA Free [NB110-55288]

Western Blot: DRP1 Antibody [NB110-55288] - Total protein from human HeLa and MCF7 cells, mouse 3T3 cells and rat PC12 cells was separated on a 7.5 % gel by SDS-PAGE, transferred to PVDF membrane and blocked in 5% non-fat milk in TBST. The membrane was probed with 2.0 ug/mL anti-DRP1 in blocking buffer and detected with an anti-rabbit HRP secondary antibody using chemiluminescence.
Immunohistochemistry-Paraffin: DRP1 Antibody - BSA Free [NB110-55288]

Immunohistochemistry-Paraffin: DRP1 Antibody - BSA Free [NB110-55288]

Immunohistochemistry-Paraffin: DRP1 Antibody [NB110-55288] - Analysis of FFPE tissue section of mouse kidney using DRP1 antibody #NB110-55288 at 1:300. The primary antibody bound to DRP1 protein in the tissue section was detected using a HRP labeled secondary antibody and DAB reagent. Nuclei of the cells were counterstained with hematoxylin. This antibody generated a diffused cytoplasmic staining of DRP1 in the epithelial cells of various tubules and in the cells of glomeruli.
Western Blot: DRP1 AntibodyBSA Free [NB110-55288]

Western Blot: DRP1 AntibodyBSA Free [NB110-55288]

Western Blot: DRP1 Antibody [NB110-55288] - Lane 1: DRP1 knockout. Lane 2: DRP1 wildtype MEFs. Stained with NB110-55288 at 1:500.
Immunocytochemistry/ Immunofluorescence: DRP1 Antibody - BSA Free [NB110-55288]

Immunocytochemistry/ Immunofluorescence: DRP1 Antibody - BSA Free [NB110-55288]

Immunocytochemistry/Immunofluorescence: DRP1 Antibody [NB110-55288] - HeLa cells were fixed for 10 minutes using 10% formalin and then permeabilized for 5 minutes using 1X PBS + 0.5% Triton X-100. The cells were incubated with anti-DRP1 at 5 ug/mL overnight at 4C and detected with an anti-rabbit Dylight 488 (Green) at a 1:500 dilution. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 40X objective.
Immunohistochemistry: DRP1 Antibody - BSA Free [NB110-55288]

Immunohistochemistry: DRP1 Antibody - BSA Free [NB110-55288]

Immunohistochemistry: DRP1 Antibody [NB110-55288] - Staining of renal tubular epithelium and visceral epithelial cells of the glomerulus. Human kidney cortex, 40X magnification.
Western Blot: DRP1 AntibodyBSA Free [NB110-55288]

Western Blot: DRP1 AntibodyBSA Free [NB110-55288]

DRP1-Antibody-Western-Blot-NB110-55288-img0019.jpg
Immunocytochemistry/ Immunofluorescence: DRP1 Antibody - BSA Free [NB110-55288]

Immunocytochemistry/ Immunofluorescence: DRP1 Antibody - BSA Free [NB110-55288]

Immunocytochemistry/Immunofluorescence: DRP1 Antibody [NB110-55288] - PC12 cells were fixed for 10 minutes using 10% formalin and then permeabilized for 5 minutes using 1X PBS + 0.05% Triton-X100. The cells were incubated with anti-DRP1 Antibody at 2 ug/ml overnight at 4C and detected with an anti-rabbit Dylight 488 (Green) at a 1:500 dilution. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 40X objective.
Flow Cytometry: DRP1 Antibody - BSA Free [NB110-55288]

Flow Cytometry: DRP1 Antibody - BSA Free [NB110-55288]

Flow Cytometry: DRP1 Antibody [NB110-55288] - An intracellular stain was performed on HeLa cells with DRP1 Antibody NB110-55288AF488 (blue) and a matched isotype control (orange). Cells were fixed with 4% PFA and then permeabilized with 0.1% saponin. Cells were incubated in an antibody dilution of 5 ug/mL for 30 minutes at room temperature. Both antibodies were conjugated to Alexa Fluor 488.
Western Blot: DRP1 AntibodyBSA Free [NB110-55288]

Western Blot: DRP1 AntibodyBSA Free [NB110-55288]

Western Blot: DRP1 Antibody [NB110-55288] - Rat spinal cord-DRP1 (81kda). Image from verified customer review.
Immunocytochemistry/ Immunofluorescence: DRP1 Antibody - BSA Free [NB110-55288]

Immunocytochemistry/ Immunofluorescence: DRP1 Antibody - BSA Free [NB110-55288]

Immunocytochemistry/Immunofluorescence: DRP1 Antibody [NB110-55288] - HeLa cells were fixed for 10 minutes using 10% formalin and then permeabilized for 5 minutes using 1X TBS + 0.5% Triton X-100. The cells were incubated with anti-DRP1 at 5.0 ug/mL overnight at 4C and detected with an anti-rabbit Dylight 488 (Green) at 1:500. Alpha tubulin (DM1A) NB100-690 was used as a co-stain at 1:1000 and detected with an anti-mouse Dylight 550 (Red) at 1:500. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 40X objective.
Immunocytochemistry/ Immunofluorescence: DRP1 Antibody - BSA Free [NB110-55288]

Immunocytochemistry/ Immunofluorescence: DRP1 Antibody - BSA Free [NB110-55288]

Immunocytochemistry/Immunofluorescence: DRP1 Antibody [NB110-55288] - NIH3T3 cells were fixed for 10 minutes using 10% formalin and then permeabilized for 5 minutes using 1X PBS + 0.05% Triton-X100. The cells were incubated with anti-DRP1 Antibody at 2 ug/ml overnight at 4C and detected with an anti-rabbit Dylight 488 (Green) at a 1:500 dilution. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 40X objective.
Flow Cytometry: DRP1 Antibody - BSA Free [NB110-55288]

Flow Cytometry: DRP1 Antibody - BSA Free [NB110-55288]

Flow Cytometry: DRP1 Antibody [NB110-55288] - An intracellular stain was performed on HeLa cells with NB110-55288 and a matched isotype control. Cells were fixed with 4% PFA and then permeablized with 0.1% saponin. Cells were incubated in an antibody dilution of 2.5 ug/mL for 30 minutes at room temperature, followed by Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Dylight 550.
Simple Western: DRP1 AntibodyBSA Free [NB110-55288]

Simple Western: DRP1 AntibodyBSA Free [NB110-55288]

Simple Western: DRP1 Antibody [NB110-55288] - Image shows a specific band for DUX4 in 0.5 mg/mL of HeLa lysate. This experiment was performed under reducing conditions using the 12-230 kDa separation system.
Simple Western: DRP1 AntibodyBSA Free [NB110-55288]

Simple Western: DRP1 AntibodyBSA Free [NB110-55288]

Simple Western: DRP1 Antibody - BSA Free [NB110-55288] - Image shows a specific band for DRP1 in 0.1 ug/uL mouse hippocampus tissue lysate. Primary antibody dilution: 1:200. Image from verified customer review.
DRP1 Antibody - BSA Free

Western Blot: DRP1 Antibody - BSA Free [NB110-55288] -

Assessment of skeletal muscle mitochondrial dynamic markers. (A) Total Drp1 protein content, (B) phospho-Drp1Ser616 protein content, (C) percent difference in total and phospho-Drp1Ser616 in ET participants relative to SED participants, (D) total Mfn1 protein content, (E) total Mfn2 protein content. (F) Representative western blots. Significant main effect of training: #, p < 0.05; ##, p < 0.01. Significant post-hoc training effect: *, p < 0.05; **, p < 0.01. Data are presented as mean ± SEM.
DRP1 Antibody - BSA Free

Detection of DRP1 in SJCRH30 Human Cell Line by Flow Cytometry.

An intracellular stain was performed on SJCRH30 human Rhabdomyosarcoma cell line with Rabbit anti-DRP1 Affinity-purified Polyclonal Antibody conjugated to FITC (Catalog # NB110-55288F, blue histogram) or matched control antibody (NBP2-24892, orange histogram) at 5 µg/mL for 30 minutes at RT.

Applications for DRP1 Antibody - BSA Free

Application
Recommended Usage

Flow (Intracellular)

1.0 ug/ml

Flow Cytometry

1:1000

Immunocytochemistry/ Immunofluorescence

1:500

Immunohistochemistry

2.5 ug/ml

Immunohistochemistry-Paraffin

2.5 ug/ml

Immunoprecipitation

reported in scientific literature

Simple Western

1:50

Western Blot

1:500
Application Notes

In Western blot a band is seen at ~81 kDa.

In Simple Western only 10 - 15 uL of the recommended dilution is used per data point.
See Simple Western Antibody Database for Simple Western validation: Tested in Human Brain lysate 0.5 mg/mL, separated by Size, antibody dilution of 1:50, apparent MW was 89 kDa. Separated by Size-Wes, Sally Sue/Peggy Sue.
The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Reviewed Applications

Read 4 reviews rated 5 using NB110-55288 in the following applications:

Flow Cytometry Panel Builder

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Advanced Features

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Flow Cytometry

Formulation, Preparation, and Storage

Purification

Immunogen affinity purified

Formulation

PBS

Format

BSA Free

Preservative

0.02% Sodium Azide

Concentration

1 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Aliquot and store at -20C or -80C. Avoid freeze-thaw cycles.

Background: DRP1

DRP1 (dynamin-related protein 1) is a highly conserved large GTPase protein which implicates in several important structural features of mitochondria, including mitochondrial shape, size, distribution, remodeling and maintenance. It functions in mitochondrial as well as peroxisomal division, and facilitates membrane fission via oligomerization into ring-like structures that wrap around fission site to constrict/split the membranes through a GTP hydrolysis-dependent mechanism. For normal brain development, DRP1 plays an essential role by mediating developmentally-regulated apoptosis during neural tube development. It is also required for a normal rate of cytochrome c release or caspase activation during apoptosis and for mitochondrial fission during mitosis, programmed necrosis execution and also for vesicle transport. Moreover, it is associated with mitochondrial structural and functional alterations such as fragmentation, phosphorylation, SUMOylation, ubiquitination and cell death. Drp1 interacts with amyloid beta, causing abnormal mitochondrial dynamics and neuronal damage in Alzheimer's disease patients, and defective DRP1 is also linked to encephalopathy, lethal, due to defective mitochondrial and peroxisomal fission (EMPF).

Long Name

Dynamin-1-like protein

Alternate Names

DLP1, DNM1L, DVLP, Dymple, HdynIV

Entrez Gene IDs

10059 (Human); 74006 (Mouse); 114114 (Rat)

Gene Symbol

DNM1L

UniProt

O00429 (Human)

Additional DRP1 Products

Product Documents for DRP1 Antibody - BSA Free

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot or batch number in the search box below.

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Product Specific Notices for DRP1 Antibody - BSA Free

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Citations for DRP1 Antibody - BSA Free

Customer Reviews for DRP1 Antibody - BSA Free (4)

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Customer Images

Showing  1 - 4 of 4 reviews Showing All
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  • DRP1 Antibody - BSA Free
    Name: Celeste Wu
    Application: Simple Western
    Sample Tested: Brain (hippocampus) tissue
    Species: Mouse
    Verified Customer | Posted 02/06/2023
    0.1ug/ul hippocampus tissue lysate with DRP1 antibody (1:200)
    DRP1 Antibody - BSA Free NB110-55288
  • DRP1 Antibody
    Name: nitesh sharma
    Application: Western Blot
    Sample Tested: Jurkat cell line
    Species: Human
    Verified Customer | Posted 10/05/2018
    DRP1 Antibody - BSA Free NB110-55288
  • DRP1 Antibody
    Name: Anonymous
    Application: Western Blot
    Sample Tested: PC12 whole cell lysate
    Species: Other
    Verified Customer | Posted 06/09/2014
  • Name: Hyun Yi
    Application: Western Blot
    Sample Tested: Rat
    Species: Rat
    Verified Customer | Posted 04/25/2014
    rat spinal cord-DRP1(81kda)
    DRP1 Antibody - BSA Free NB110-55288

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Showing  1 - 4 of 4 reviews Showing All

Protocols

View specific protocols for DRP1 Antibody - BSA Free (NB110-55288):

Protocol for Flow Cytometry Intracellular Staining
Sample Preparation.
1. Grow cells to 60-85% confluency. Flow cytometry requires between 2 x 105 and 1 x 106 cells for optimal performance.
2. If cells are adherent, harvest gently by washing once with staining buffer and then scraping. Avoid using trypsin as this can disrupt certain epitopes of interest. If enzymatic harvest is required, use Accutase, Collagenase, or TrypLE Express for a less damaging option.
3. Reserve 100 uL for counting, then transfer cell volume into a 50 mL conical tube and centrifuge for 8 minutes at 400 RCF.
a. Count cells using a hemocytometer and a 1:1 trypan blue exclusion stain to determine cell viability before starting the flow protocol. If cells appear blue, do not proceed.
4. Re-suspend cells to a concentration of 1 x 106 cells/mL in staining buffer (NBP2-26247).
5. Aliquot out 100 uL samples in accordance with your experimental samples.

Tip: When cell surface and intracellular staining are required in the same sample, it is advisable that the cell surface staining be performed first since the fixation and permeabilization steps might reduce the availability of surface antigens.

Intracellular Staining.
Tip: When performing intracellular staining, it is important to use appropriate fixation and permeabilization reagents based upon the target and its subcellular location. Generally, our Intracellular Flow Assay Kit (NBP2-29450) is a good place to start as it contains an optimized combination of reagents for intracellular staining as well as an inhibitor of intracellular protein transport (necessary if staining secreted proteins). Certain targets may require more gentle or transient permeabilization protocols such as the commonly employed methanol or saponin-based methods.
Protocol for Cytoplasmic Targets:
1. Fix the cells by adding 100 uL fixation solution (such as 4% PFA) to each sample for 10-15 minutes.
2. Permeabilize cells by adding 100 uL of a permeabilization buffer to every 1 x 106 cells present in the sample. Mix well and incubate at room temperature for 15 minutes.
a. For cytoplasmic targets, use a gentle permeabilization solution such as 1X PBS + 0.5% Saponin or 1X PBS + 0.5% Tween-20.
b. To maintain the permeabilized state throughout your experiment, use staining buffer + 0.1% of the permeabilization reagent (i.e. 0.1% Tween-20 or 0.1% Saponin).
3. Following the 15 minute incubation, add 2 mL of the staining buffer + 0.1% permeabilizer to each sample.
4. Centrifuge for 1 minute at 400 RCF.
5. Discard supernatant and re-suspend in 100 uL of staining buffer + 0.1% permeabilizer.
6. Add appropriate amount of each antibody (eg. 1 test or 1 ug per sample, as experimentally determined).
7. Mix well and incubate at room temperature for 30 minutes- 1 hour. Gently mix samples every 10-15 minutes.
8. Following the primary/conjugate incubation, add 1-2 mL/sample of staining buffer +0.1% permeabilizer and centrifuge for 1 minute at 400 RCF.
9. Wash twice by re-suspending cells in staining buffer (2 mL for tubes or 200 uL for wells) and centrifuging at 400 RCF for 5 minutes. Discard supernatant.
10. Add appropriate amount of secondary antibody (as experimentally determined) to each sample.
11. Incubate at room temperature in dark for 20 minutes.
12. Add 1-2 mL of staining buffer and centrifuge at 400 RCF for 1 minute and discard supernatant.
13. Wash twice by re-suspending cells in staining buffer (2 mL for tubes or 200 uL for wells) and centrifuging at 400 RCF for 5 minutes. Discard supernatant.
14. Resuspend in an appropriate volume of staining buffer (usually 500 uL per sample) and proceed with analysis on your flow cytometer.


Immunocytochemistry Protocol

Culture cells to appropriate density in 35 mm culture dishes or 6-well plates.

1. Remove culture medium and add 10% formalin to the dish. Fix at room temperature for 30 minutes.
2. Remove the formalin and add ice cold methanol. Incubate for 5-10 minutes.
3. Remove methanol and add washing solution (i.e. PBS). Be sure to not let the specimen dry out. Wash three times for 10 minutes.
4. To block nonspecific antibody binding incubate in 10% normal goat serum from 1 hour to overnight at room temperature.
5. Add primary antibody at appropriate dilution and incubate at room temperature from 2 hours to overnight at room temperature.
6. Remove primary antibody and replace with washing solution. Wash three times for 10 minutes.
7. Add secondary antibody at appropriate dilution. Incubate for 1 hour at room temperature.
8. Remove antibody and replace with wash solution, then wash for 10 minutes. Add Hoechst 33258 to wash solution at 1:25,0000 and incubate for 10 minutes. Wash a third time for 10 minutes.
9. Cells can be viewed directly after washing. The plates can also be stored in PBS containing Azide covered in Parafilm (TM). Cells can also be cover-slipped using Fluoromount, with appropriate sealing.

*The above information is only intended as a guide. The researcher should determine what protocol best meets their needs. Please follow safe laboratory procedures.

Immunohistochemistry-Paraffin Embedded Sections

Antigen Unmasking:
Bring slides to a boil in 10 mM sodium citrate buffer (pH 6.0) then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench-top for 30 minutes (keep slides in the sodium citrate buffer all the time).

Staining:
1. Wash sections in deionized water three times for 5 minutes each.
2. Wash sections in PBS for 5 minutes.
3. Block each section with 100-400 ul blocking solution (1% BSA in PBS) for 1 hour at room temperature.
4. Remove blocking solution and add 100-400 ul diluted primary antibody. Incubate overnight at 4 C.
5. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
6. Add 100-400 ul HRP polymer conjugated secondary antibody. Incubate 30 minutes at room temperature.
7. Wash sections three times in wash buffer for 5 minutes each.
8. Add 100-400 ul DAB substrate to each section and monitor staining closely.
9. As soon as the sections develop, immerse slides in deionized water.
10. Counterstain sections in hematoxylin.
11. Wash sections in deionized water two times for 5 minutes each.
12. Dehydrate sections.
13. Mount coverslips.



Western Blot Protocol

1. Perform SDS-PAGE (4-12%) on samples to be analyzed, loading 30 ug of total protein per lane.
2. Transfer proteins to Nitrocellulose according to the instructions provided by the manufacturer of the transfer
apparatus.
3. Rinse membrane with dH2O and then stain the blot using Ponceau S for 1-2 minutes to access the transfer of proteins onto the nitrocellulose membrane. Rinse the blot in water to remove excess stain and mark the lane locations and locations of molecular weight markers using a pencil.
4. Rinse the blot in TBS for approximately 5 minutes.
5. Block the membrane using 5% non-fat dry milk + 1% BSA in TBS, 1 hour at room temperature.
6. Rinse the membrane in dH2O and then wash the membrane in wash buffer [TBS + 0.1% Tween] 3 times for 10 minutes each.
7. Dilute the rabbit anti-DRP1 primary antibody (NB 110-55288) in blocking buffer and incubate 2 hours at room temperature.
8. Rinse the membrane in dH2O and then wash the membrane in wash buffer [TBS + 0.1% Tween] 3 times for 10 minutes each.
9. Apply the diluted rabbit-IgG HRP-conjugated secondary antibody in blocking buffer (as per manufacturers
instructions) and incubate 1 hour at room temperature.
10. Wash the blot in wash buffer [TBS + 0.1% Tween] 3 times for 10 minutes each (this step can be repeated as required to reduce background).
11. Apply the detection reagent of choice in accordance with the manufacturers instructions (Pierce, ECL).

Note: Tween-20 can be added to the blocking or antibody diultion buffer at a final concentration of 0.05-0.2%, provided it does not interfere with antibody-antigen binding.

IHC-FFPE sectionsI. Deparaffinization:

A. Treat slides with Xylene: 3 changes for 5 minutes each. Drain slides for 10 seconds between changes.
B. Treat slides with 100% Reagent Alcohol: 3 changes for 5 minutes each. Drain slides for 10 seconds between changes.

II. Quench Endogenous Peroxidase:

A.Place slides in peroxidase quenching solution: 15-30 minutes.

To Prepare 200 ml of Quenching Solution:

-Add 3 ml of 30% Hydrogen Peroxide to 200 ml of Methanol.
-Use within 4 hours of preparation

B. Place slides in distilled water: 2 changes for 2 minutes each.

III. Retrieve Epitopes:

A. Preheat Citrate Buffer. Place 200 ml of Citrate Buffer Working Solution into container, cover and place into steamer. Heat to 90-96 degrees Celcius.
B. Place rack of slides into hot Citrate Buffer for 20 minutes. Cover.
C.Carefully remove container with slides from steamer and cool on bench, uncovered, for 20 minutes.
D. Slowly add distilled water to further cool for 5 minutes.
E. Rinse slides with distilled water. 2 changes for 2 minutes each.

IV. Immunostaining Procedure:

A. Remove each slide from rack and circle tissue section with a hydrophobic barrier pen (e.g. Liquid Blocker-Super Pap-Pen).
B. Flood slide with Wash Solution. Do not allow tissue sections to dry for the rest of the procedure.
C. Drain wash solution and apply 4 drops of Blocking Reagent to each slide and incubate for 15 minutes.
D. Drain Blocking Reagent (do not wash off the Blocking Reagent), apply 200 ul of Primary Antibody solution to each slide, and incubate for 1 hour.
E. Wash slides with Wash Solution: 3 changes for 5 minutes each.
F. Drain wash solution, apply 4 drops of Secondary antibody to each slide and incubate for 1 hour.
G. Wash slides with Wash Solution: 3 changes for 5 minutes each.
H. Drain wash solution, apply 4 drops of DAB Substrate to each slide and develop for 5-10 minutes. Check development with microscope.
I. Wash slides with Wash Solution: 3 changes for 5 minutes each.Wash slides with Wash Solution: 3 changes for 5 minutes each
J. Drain wash solution, apply 4 drops of Hematoxylin to each slide and stain for 1-3 minutes. Increase time if darker counterstaining is desired.
K. Wash slides with Wash Solution: 2-3 changes for 2 minutes each.
L. Drain wash solution and apply 4 drops of Bluing Solution to each slide for 1-2 minutes.
M. Rinse slides in distilled water.
N. Soak slides in 70% reagent alcohol: 3 minutes with intermittent agitation.
O. Soak slides in 95% reagent alcohol: 2 changes for 3 minutes each with intermittent agitation.
P. Soak slides in 100% reagent alcohol: 3 changes for 3 minutes each with intermittent agitation. Drain slides for 10 seconds between each change.
Q. Soak slides in Xylene: 3 changes for 3 minutes each with intermittent agitation. Drain slides for 10 seconds between each change.
R. Apply 2-3 drops of non-aqueous mounting media to each slide and mount coverslip.
S. Lay slides on a flat surface to dry prior to viewing under microscope.

NOTES:

-Use treated slides (e.g. HistoBond) to assure adherence of FFPE sections to slide.
-Prior to deparaffinization, heat slides overnight in a 60 degrees Celcius oven.
-All steps in which Xylene is used should be performed in a fume hood.
-For Epitope Retrieval, a microwave or pressure cooker may be substituted for the steamer method. Adjust times as necessary depending on conditions.
-For the initial IHC run with a new primary antibody, test tissues with and without Epitope Retrieval. In some instances, Epitope Retrieval may not be necessary.
-200 ul is the recommended maximum volume to apply to a slide for full coverage. Using more than 200 ul may allow solutions to wick off the slide and create drying artifacts. For small tissue sections less than 200 ul may be used.
-5 minutes of development with DAB Substrate should be sufficient. Do not develop for more than 10 minutes. If 5 minutes of development causes background staining, further dilution of the primary antibody may be necessary.
-Hematoxylin should produce a light nuclear counterstain so as not to obscure the DAB staining. Counterstain for 1-1.5 minutes for nuclear antigens. Counterstain for 2-3 minutes for cytoplasmic and membranous antigens. If darker counterstaining is desired increase time (up to 10 minutes).

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