entangles free floating tissue

[skumarmadhu]

Dear friends
I am new to IHC, i perfuse mice brain( 7day pups) fix in PFA for 24h in PFA, then transfer to 1X PBS, embedd in 3% agarose, take 40 micron free floating sections in 1XPBS
I wash them in PBS
Permialize in 3%BSA,0.1%triton-X100 (5min)
Block them in 1%BSA, 10%NGS, 0.2%milk,0.1%TritonX-100 (1hr)
Primary in the same blocking soln except NGS (overnight at 4)
secondary Alexa 488/594 in the same blocking buffer

My tissues are intially very good but till i put them in the seconary they will be entangled and looses its shape, and i will be unable to mount them properly, they will become so fragile and break up even by gentle touch, earlier i use to use 0.3% triton, but now i reduce it to 0.1% but still i have that problem, plz can anyone find soln and help as soon as possible, i will be greatful,
Madhu