Background or unspecific staining often occurs for IHC antibody staining. Here we list commonly used blocking solutions during antibody staining procedure to reduce background or unspecific staining.
Peroxidase Blocking Soluiton (3% H2O2 in PBS):
30% H2O2 ------------------------- 10 ml
1X PBS ----------------------------- 90 ml
Mix well and store at 4 ºC for up to 3 months.
Block sections for 10 minutes before blocking step or after primary antibody incubation. Note: this solution is recommended for paraffin sections.
Peroxidase Blocking Soluiton (0.3% H2O2 in Methanol):
30% H2O2 ------------------------- 1 ml
Methanol -------------------------- 99 ml
Mix well and store at 4 ºC.
Block sections for 20-30 minutes before serum blocking step prior to primary antibody incubation. Note: this solution is recommended for frozen sections. Blocking after primary antibody incubation may cause tissue damage.
Avidin/Biotin Block:
Avidin 0.001% in PBS
Biotin 0.001% in PBS
Store these blocking solution at 4 ºC.
Incubate sections for 10-15 min each and rinse with PBS between steps. Recommended to block before primary antibody incubation.
Normal Goat Serum Blocking Solution:
2% goat serum (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)
0.01M PBS, pH 7.2
Mix well and store at 4 ºC.
Normal Horse Serum Blocking Solution:
2% horse serum (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)
0.01M PBS, pH 7.2
Mix and store at 4 ºC.
Normal Swine Serum Block Solution:
2% swine serum (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)
0.01M PBS, pH 7.2
Mix and store at 4 ºC.
Normal Donkey Serum Block Solution:
2% donkey serum (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)
0.01M PBS, pH 7.2
Mix and store at 4 ºC.
Normal Rabbit Serum Block Solution:
2% rabbit serum (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)
0.01M PBS, pH 7.2
Mix and store at 4 ºC.
Universal Blocking Buffer:
1%BSA (blocking & stabilizer)
0.1% cold fish skin gelatin (blocking)
0.5% Triton X-100 (penetration enhancer)
0.05% sodium azide (preservative)
0.01M PBS, pH 7.2-7.4
Mix well and store at 4 ºC.
Note: this blocking buffer can also used as un-conjugated primary antibody dilution buffer. Do not use it to dilute HRP conjugated antibody as the sodium azide is inhibitor of HRP. Do not use it to dilute Alkaline Phosphatase (AP) conjugated antibody as the phosphate in PBS is an inhibitor of AP.