PROBLEM WITH IHC PROTOCOL

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prashantsoni
prashantsoni's picture
PROBLEM WITH IHC PROTOCOL

 Dear all

I am doing  IHC on rat heart sections using goat primary antibody for collagen 1, 3 & TGF beta with secondary antibody antigoat HRP conjugated and DAB as a substrate. I am using the primary and secondary Ab at the conc. of 1:200. I m not gettin any kind of signals although my cunterstaining is good and there is no background stain.
I have few querries:

Since I m using Abs generated in goat can i use 5 % normal goat serum for blocking. Currently I m using 3% BSA as a blockinf agent.

Ours vecta stain kit is for mouse and rabbit secondary Abs. Can I use them with my Abs which are generated in goat.

If cannot then what can be the alternative procedure if any

Arvind Singh Pundir
Arvind Singh Pundir's picture
 Hi Prashant,

 Hi Prashant,
      mention your detailed protocol  followed for your IHC expt. so that we can troubleshoot the exact problem 

prashantsoni
prashantsoni's picture
 Procedure for IHC:

 Procedure for IHC:
Treat 5 slides at a time with following in coupling jars unless otherwise stated:
1- Xylene= 5 min X 2
2- 100% alcohol= 5 min X 2
3- 90% alcohol= 5 min X 2
4- 80% alcohol= 5 min X 1
5- 70% alcohol= 5 min X 1
6- TBS (ph 7.6)= 5 min X 2
Put diluted Proteinase K (1:100) on sections on slides. Incubate for 20 min at room temperature.
Cover slides with a piece of parafilm coverslip

H2O2 (3%) = 10 min X 1
TBS (ph 7.6)= 5 min X 2
Gently tap off the excess liquid and carefully dry the glass slides around the specimen

Blocking with 3% BSA in TBS I m not using 5% goat serum because of goat primary Ab
Put Primary antibody solution (1:200) on sections on slides. Incubate overnight (12-18 hrs) at 4 0C in humidity chamber

Remove the primary antibody.
Fresh TBS (ph 7.6)= 5 min X 3

Put secondary antibody solution - (1:200) on sections on slides. Incubate for 2-2.30 hrs in humidity chamber at room temperature.
? Cover slides with a piece of parafilm coverslip

Remember i m not using vecta stain kit becaue i m using primary Ab generated from goat and secondary rabbit antigoat Ab

TBS (ph 7.6)= 5 min X 3
 Put diluted solution of DAB( 1:10) on sections on slides for about 3 min or (30 sec) or till a faint brown colour is observed

TBS (ph 7.6)= 5 min X 1
 Put Haematoxyline stain (Gill 2 grade) on sections on slides. Incubate for 2 min or till  blue colour is observed.

Tap water= 5 min X 1
24- Acid alcohol= Just dip and remove

Dehydration of sections by reverse procedure from 70% alcohol to xylene.
a) 70% alcohol= 5 min X 1
b) 80% alcohol= 5 min X 1
c) 90% alcohol= 5 min X 2
d) 100% alcohol= 5 min X 2
e) Xylene= 5 min X 2
Put DPX on slides around sections (not on the sections) and put coverslip on the slide without air bubble. Keep it for drying for 3-4 hrs.
Observe under leica microscope.

 

Arvind Singh Pundir
Arvind Singh Pundir's picture
 try increasing the dilution

 try increasing the dilution of your primary ab , and use serum from the species in which your secondary ab is raised, for example if you are using secondary as goat anti rbt then use goat serum as blocking and its conc may vary from 3-10% as optimal for blocking the background........