Recombinant Anti-NAPSIN A antibody [EPR6257] (ab129189)

Our antigen retrieval method used for ab92378 and ab129189 is described below.

Antigen Retrieval

This is recommended Heat Induced Epitope Retrieval (HIER) using Decloaking Chamber/Pressure Cooker. Hot water bath or Microwave with temperature sensor can be also used (protocol would vary depending on the method used).

1. Add 500 ml of dH 2O to Decloaker/Pressure Cooker.

2. Immerse slides into staining dish containing Antigen Retrieval Solution. Place staining dish into decloaking chamber.

3. Program to run for 30 seconds at 125° C, followed by 10 seconds at 90° C.

4. Let it cool down to room temperature (10 - 20 minutes).

5. Removes slides and rinse in TBST.

6. Proceed to Staining step.

Thank you fo ryour message.

To clarify regarding ab135618 Anti-PAX8 antibody, the datasheet states we sell this at 100 ug, at a concentration of 0.25 mg/ml. Therefore, you can calculate that at this concentrations, 100 ug will be in 250 ul of liquid.

For the protocols, I am pleased to provide the general testing IHC-P protocols from the originators. I have copied thesebelow. I am sorry it has taken a while to obtain these.Please note that these will be a guideline only and further individual optimizationmay be required.

As stated in the previous email, recommended dilutions in IHC-P for these antibodies are listed on the datasheets:

ab129189 NAPSIN AIHC-P: 1/100 - 1/250.
ab92378 HNF4 IHC-P: 1/100 - 1/250
ab135618 PAX8IHC-P: 1/50 - 1/100.

I hope this will be helpful to you. If you have any further questions, please do not hesitate to contact us.

Protocol for ab135618 Anti-PAX8 antibody


Preparing Tissue Sections for Immunostaining:

1. Fix the tissue in 10% formalin at 4oC overnight.
2. Embed fixed tissue in paraffin.
3. Mount tissue sections on slides.
4. Clear the paraffin with xylene for ten minutes; move slides to a fresh dish of xylene for an additional ten minutes. NOTE: Perform all xylene washes in a fume hood!
5. Rinse the slides twice for 2 minutes in 100% alcohols (18:1:1 100% ethanol: 100% methanol: 100% isopropanol).
6. Rinse the slides twice for 2 minutes in a 95% solution of the 100% alcohols.
7. Place slides in an 80% solution of the 100% alcohols for 2 minutes, followed by deionized water for 5 minutes.
8. Rinse slides several times with fresh deionized water followed by another five minutes wash using fresh water.


Sodium Citrate Antigen Retrieval:

1. Place slides in a glass slide holder and fill in the rest of the rack with blank slides (10 totals) to ensure even heating.
2. Place rack in 600 ml of 10 mM sodium citrate, pH 6.0 in a glass 2 L-beaker. Mark a line at the top of the liquid on the beaker.
3. Microwave for 20 min total, replacing evaporated water every 5 min.
4. Cool slides for 20 min.
5. Wash 4 X 3 min in ddH2O, and 3 min in 1X PBS.


Blocking


1. Block endogenous peroxidases by soaking slides in a solution of 90% methanol/3% H2O2 for 15 minutes at room temperature. Wash 3 X in PBS.
2. Immerse slides in a dish containing blocking buffer (serum from host species of secondary antibody to be used, diluted 1:10 in TBS). Incubate at 37oC for one hour.


Incubation with Primary Antibodies


1. Cover the tissue sections with primary antibody diluted in blocking buffer. Antibody is diluted 1:50 and 1:100. Incubate for 1 hour at 37oC.
2. Blot excess liquid from slides and rinse three times in PBS for five minutes each wash.
Incubation with Secondary Antibodies
3. Cover the tissue sections with secondary antibody diluted in blocking buffer according to manufacturer’s instructions. We routinely use prediluted universal secondary antibody (Jackson ImmunoResearch Laboratories). Incubate at 37oC for 30 min.
4. Blot excess liquid and rinse twice in TBS for five minutes each wash.


Counterstaining and Visualization

1. Counterstain with Hematoxylin.
2. Rinse several times in deionized water. Blot excess water around tissue, then apply one drop of mounting media to tissue and place coverslip over slide. Seal with nail polish.

Citrate Solutions:
Description: Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining for immunohistochemical detection of certain proteins. The citrate based solution is designed to break the protein cross-links, thereby unmasking the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections and enhancing the staining intensity of antibodies.


Sodium Citrate Buffer (10mM Sodium Citrate, 0.05% Tween 20, pH 6.0):

Tri-sodium citrate (dihydrate) 2.94 g
Distilled water 1000 ml
Mix to dissolve. Adjust pH to 6.0 with 1N HCl and then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4¢XC for longer storage.
Citrate Buffer (10mM Citric Acid, 0.05% Tween 20, pH 6.0):
Citric acid (anhydrous) 1.92 g
Distilled water 1000 ml
Mix to dissolve. Adjust pH to 6.0 with 1N NaOH and then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4oC for longer storage.

Washing Buffer:

1 X PBS:
NaCl 8 g
KCl 0.2 g
Na2HPO 41.44 g
KH2PO4 0.24 g
Distilled Water 800 mL
Adjust pH to 7.2 with HCl.
Adjust volume to 1 L with additional H2O


Normal Serum Blocking Buffer:
2% serum from host species of secondary antibody (blocking)
1% BSA (stabilizer)
0.1% cold fish skin gelatin (blocking)
0.1% Triton X-100 (penetration enhancer)
0.05% Tween 20 (detergent and surface tension reducer)
0.05% sodium azide (preservative)
Dissolve in 1X PBS
Mix well and store at 40C.


Avidin/Biotin Block:
Avidin 0.001% in 1 X PBS
Biotin 0.001% in 1 X PBS
Store these blocking solution at 4oC.


Primary Antibody Dilution Buffer:
1%BSA (stabilizer and blocking)
0.1% cold fish skin gelatin (blocking)
0.05% sodium azide (preservative)
0.01M PBS pH7.2

Note: 1) Antibodies diluted using this buffer can be stored at 4°C for 6 months without reducing binding activity. 2) This buffer cannot be used for diluting HRP conjugated antibodies since sodium azide is an inhibitor of HRP.


Peroxidase Blocking Solution (3% H2O2 in PBS):
30% H2O2 2 ml
1 X PBS - 18 ml
Mix well and store at 4°C for up to 3 months.
This solution is recommended for paraffin sections


References:
1. Shi SR, Chaiwun B, Young L, Cote RJ, Taylor CR. Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections. J Histochem Cytochem. 1993 Nov; 41(11):1599-604. PubMed Abstract
2. Kanai K, Nunoya T, Shibuya K, Nakamura T, Tajima M (1998) Variations in effectiveness of antigen retrieval pretreatments for diagnostic immunohistochemistry. Res Vet Sci. 64(1):57-61. PubMed Abstract
3. Brown RW, Chirala R (1995) Utility of microwave-citrate antigen retrieval in diagnostic immunohistochemistry. Mod Pathol. 8(5):515-20. PubMed Abstract
4. Morgan JM, Navabi H, Schmid KW, Jasani B. Possible role of tissue-bound calcium ions in citrate-mediated high-temperature antigen retrieval. J Pathol. 1994 Dec; 174(4):301-7. PubMed Abstract
5. Pellicer EM, Sundblad A (1994) Antigen retrieval by microwave oven with buffer of citric acid. Medicina (B Aires). 54(2):129-32. PubMed Abstract
6. Shi SR, Chaiwun B, Young L, Cote RJ, Taylor CR (1993) Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections. J Histochem Cytochem. 41(11):1599-604. PubMed Abstract
7. Brown, D., et al. (1996) Antigen retrieval in cryostat tissue sections and cultured cells by treatment with sodium dodecyl sulfate (SDS).Histochem Cell Biol 105:261–267.



Protocols for ab92378 Anti-HNF4 antibody [EPR3648] and ab129189 Anti-NAPSIN A antibody [EPR6257]:


1. Solutions and reagents

1.1 Xylene
1.2. Ethanol, anhydrous denatured, histological grade (100%, 95%, 70%, 50%)
1.3. Washing buffer/TBST: 1X TBS/0.1% Tween-20, pH to 7.6.
1.4. Distilled water (dH 2O)
1.5. Antigen Retrieval Solution: 0.01M Sodium Citrate Buffer, pH 6.0
To prepare Antigen Retrieval stock solutions:

10X Stock: Dissolve 29.4 g sodium citrate trisodium salt dihydrate (C 6H 5Na 3O 7 2H 2O)
in 1 liter of dH2O. Add 5mL Tween-20.

1X Working Solution: Mix 200mL 10X stock with 1800mL dH2O; pH to 6.0

1.6. 3% Hydrogen Peroxide
1.7. Blocking Buffer: 10% serum in PBS (serum origin depends on the host of the secondary antibody)
1.8. Primary Antibody Diluent: 5% serum in PBS (serum origin depends on the host of the secondary antibody)
1.9. Hematoxylin
1.10. Permanent Mounting Medium



2. IHC Protocol

2.1. Deparaffinization/Rehydration
2.1.1. Heat slides in an oven at 65 °C for 1 hour.
2.1.2. De-paraffinize/hydrate using the following series of washes: two Xylene washes (3 min each), followed by two 100% ethanol rinses (3 min each), followed by 95% ethanol, 70% ethanol, 50% ethanol, 30% ethanol, followed by TBST wash for 3 min on a shaker.


2.2. Antigen Retrieval

This is recommended Heat Induced Epitope Retrieval (HIER) using Decloaking Chamber/Pressure Cooker. Hot water bath or Microwave with temperature sensor can be also used (protocol would vary depending on the method used).

2.2.1. Add 500 ml of dH 2O to Decloaker/Pressure Cooker.
2.2.2. Immerse slides into staining dish containing Antigen Retrieval Solution. Place staining dish into decloaking chamber.
2.2.3. Program to run for 30 seconds at 125° C, followed by 10 seconds at 90° C.
2.2.4. Let it cool down to room temperature (10 - 20 minutes).
2.2.5. Removes slides and rinse in TBST.
2.2.6. Proceed to Staining step.


2.3. Staining

2.3.1. Wash slides with TBST for 3 min on a shaker.
2.3.2. Inactivate endogenous peroxidase by covering tissue with 3% hydrogen peroxide for 5 min.
2.3.3. Wash slides three times with TBST (3 min each on a shaker).
2.3.4. Block slides with the blocking solution for 1 hour.
2.3.5. Dilute primary antibody in primary antibody diluent per recommendation on data sheet.
2.3.6. Apply primary antibody to each section and incubate overnight in the humidified
chamber (4 °C).
2.3.7. Wash slides three times with TBST (3 min each on a shaker).
2.3.8. Apply to each section secondary HRP-conjugated anti-rabbit antibody diluted in the blocking solution per manufacturer's recommendation; incubate for 30 min at room temperature.
2.3.9. Wash slides three times with TBST (5 min each on a shaker).
2.3.10. Add freshly prepared DAB substrate to the sections and incubate until stain develops (generally 1 min).
2.3.11. Rinse sections with water.
2.3.12. Counterstain with Hematoxylin (generally 10 seconds).
2.3.13. Rinse sections with water.
2.3.14. Dehydrate samples using two washes with 100% Ethanol (3 min each), followed by two rinses with Xylene (3 min each).

2.3.15. Mount coverslips on slides using permanent mounting medium.

Thank you for your message which has been forwarded to the scientific support team.

I am sorry it is difficult to let you know the amount of antibody required for one analysis. This will be very individual and will depend on:

1. The dilution of antibody used, which will need to be optimized by the end user.

2. The amount of antibody solution added to each sample.

3. The application being used.

I can suggest to use the following information from the online datasheets as a guide. This provides the amount of antibody provided in each vial and also the recommended dilutions, which will require optimization for individual experiments:

ab129189 NAPSIN A 100 µl
Recommended dilutions:
WB: 1/1000 - 1/10000.
IHC-P: 1/100 - 1/250.


ab92378 HNF4 100 µl
Reommended dilutions:
WB: 1/1000 - 1/10000
IP: 1/10 - 1/100.
IHC-P: 1/100 - 1/250
ICC: 1/100 - 1/250.
Flow Cyt: 1/100.

ab135618 PAX8 250 ul
Recomended dilutions.
WB: 1/100 - 1/500.
IHC-P: 1/50 - 1/100.
Flow Cyt: 1/10 - 1/50.

I hope this will be helpful to you. If you require any further assistance, please let me know which application you are using, and how much antibody solution you will use for each application. If you have any further questions, please do not hesitate to let me know.