Question (48698) | Anti-HCN4 antibody [SHG 1E5] (ab32675)

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Question



) Abcam product code ab 32675 (HCN4)

2) Abcam order reference number or product batch number: LotNr GR75526-1

3) Description of the problem : No staining at all

4) Sample preparation:
Species
Type of sample: PFA/formalin fixed paraffin embedded sections,:
Sample preparation: Formalin fixed paraffin embedded
Positive control human braun and human heart
Negative control: without primaty antibody

5) Fixation step
Yes
If yes: Fixative agent and concentration: 10% buffered Formalin
Fixation time: 5h
Fixation temperature: RT

6) Antigen retrieval method: Tris EDTA pH: 9 and Citrate pH:6

7) Permeabilization method:
Did you do a permeabilization step (details please) or add permeabilizing agent in any dilution buffers? Tried with and without
Permeabilizing agent and concentration: 1% triton-x100 in PBS


8) Blocking agent (eg BSA, serum…): goat serum
Concentration: 2%
Blocking time : 30 min
Blocking temperature : RT

9) Endogenous peroxidases blocked? Yes with methanol and H2O2
Endogenous biotins blocked? No?

10) Primary antibody (If more than one was used, describe in “additional notes”) :
Concentration or dilution : 1:50, 1:100; 1:200; 1:500
Diluent buffer PBS with BSA
Incubation time Over night 4°C

11) Secondary antibody: anti-rat igG biotylinalted (VECTOR)
Species: rabbit
Reacts against: rat IgG
Concentration or dilution : 1:500
Diluent buffer : PBS with BSA
Incubation time 30 min
Fluorochrome or enzyme conjugate: biotynilated (ABC kit HRP-coppeled see below at 13)

12) Washing after primary and secondary antibodies:
Buffer PBS + 0,05% tween
Number of washes 3 times 5 min

13) Detection method
ABC elite kit for peroxidase substrate AND Vector VIP Peroxidase substrate Pink

14) How many times have you run this staining? 3times
Do you obtain the same results every time? Yes
What steps have you altered to try and optimize the use of this antibody?
Different Antibody concentrations 1:50, 1:100; 1:200; 1:500,
tried with and wthout permeabilisation; tried with Alexa-Fluor coppeld secondary antibody (IF)

Answer

Es tut mir leid, dass Sie Probleme mit unserem Antikörper hatten.

Ich habe da noch ein zwei Fragen bezüglich des Protokolls:

1. Wie lange haben Sie die Antigen Maskierung durchgeführt? Haben Sie eine Zeitreihe durchgeführt?

2. Wann und wie lange haben Sie die endogene Peroxidase geblockt?

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